Anti-pacap antibodies and uses thereof

ABSTRACT

The present invention is directed to antagonistic antibodies and antigen binding fragments thereof having binding specificity for PACAP. These antibodies inhibit, block or neutralize at least one biological effect associated with PACAP, e.g., vasodilation. In exemplary embodiments these antibodies and antigen binding fragments thereof may comprise specific V H , V L , and CDR polypeptides described herein. In some embodiments these antibodies and antigen binding fragments thereof bind to and/or compete for binding to specific epitope(s) on human PACAP. The invention is further directed to using these antagonistic anti-PACAP antibodies, and binding fragments thereof, for the diagnosis, assessment, and treatment of diseases and disorders associated with PACAP and conditions where antagonism of PACAP-related activities, such as vasodilation, mast cell degranulation, and/or neuronal activation, are therapeutically beneficial, e.g., headache and migraine indications.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 62/148,550, filed Apr. 16, 2015, U.S. Provisional Application Ser.No. 62/148,557, filed Apr. 16, 2015, U.S. Provisional Application Ser.No. 62/148,562, filed Apr. 16, 2015, U.S. Provisional Application Ser.No. 62/148,596, filed Apr. 16, 2015, U.S. Provisional Application Ser.No. 62/148,643, filed Apr. 16, 2015, U.S. Provisional Application Ser.No. 62/148,583, filed Apr. 16, 2015, U.S. Provisional Application Ser.No. 62/148,640, filed Apr. 16, 2015, each of which is herebyincorporated by reference in its entirety.

SEQUENCE DISCLOSURE

This application includes as part of its disclosure an electronicsequence listing text file named “43257o5809.txt”, having a size of165,209 bytes and created on Feb. 29, 2016, which is hereby incorporatedin its entirety.

FIELD OF THE INVENTION

This invention generally pertains to antibodies and antigen bindingfragments thereof, preferably humanized, chimerized, and humanantibodies and antigen binding fragments thereof, and compositionscontaining such antibodies and antigen binding fragments thereof,wherein such antibodies and antigen binding fragments thereofspecifically bind to Pituitary Adenylate Cyclase-Activating Polypeptide(“PACAP”) and therapeutic and diagnostic uses for the antibodies,antigen binding fragments, and compositions thereof.

BACKGROUND

Pituitary Adenylate Cyclase-Activating Polypeptide (“PACAP”) is a memberof the secretin/vasoactive intestinal peptide (“VIP”)/growthhormone-releasing hormone (“GHRH”) family. PACAP is a multifunctionalvasodilatory peptide that exists in two α-amidated active forms, onewith 38 amino acids (PACAP38; SEQ ID NO: 1241) and the other with 27amino acids (PACAP27; SEQ ID NO: 1242). Both peptides have the sameN-terminal 27 amino acids and are synthesized from the same precursorprotein, preproPACAP (See, Moody et al., Curr. Opin. Endocrinol.Diabetes Obes., 18(1):61-67 2011). PACAP38 is the more prevalent activeform, representing up to 90% of PACAP forms in mammalian tissues (See.Kaiser and Russo, Neuropeptides, 47:451-461 2013). The sequence ofPACAP38 is identical in all mammals and differs from the avian andamphibian orthologs by only one amino acid (See, Vaudry et al.,Pharmacol. Rev., 52:269-324 2000). The secretin/VIP/GHRH family includesmammalian peptide histidine methioneamide (“PHM”), secretin, glucagon,glucagon-like peptide-1 (“GLP1”), glucagon-like peptide-2 (“GLP2”),glucose-dependent-insulinotrophic-polypeptide (“GIP”), andgrowth-hormone-releasing-factor (“GRF”). PACAP27 has 68% sequenceidentity to VIP at the amino acid level (See, Vaudry et al. 2000).

PACAP is widely distributed in the brain and peripheral organs, e.g.,the endocrine system, gonads, sympathetic neurons, respiratory system,gastrointestinal tract, cardiovascular system, and urogenital tracts(See, Schytz et al., Neurotherapeutics, 7:191-196 2010). In particular,PACAP is expressed throughout the nervous system, including a presencein the trigeminovascular system, trigeminal ganglia, spinal cord,hypothalamus, and pituitary. PACAP has roles in neurodevelopment,neuroprotection, neuromodulation, neurogenic inflammation, andnociception with multiple actions (See, Kaiser and Russo 2013).

Consistent with its widespread distribution, PACAP exerts pleiotropiceffects including modulation of neurotransmitter release, vasodilation,bronchodilation, and activation of intestinal motility, increase ofinsulin and histamine secretion, as well as stimulation of cellproliferation and/or differentiation. PACAP has been shown to act as ahormone, a neurohormone, a neurotransmitter, and a trophic factor in anumber of tissues (Vaudry et al., Pharmacological Rev., 52(2):269-324,2000).

The biological effects of PACAP are mediated via three differentG-protein coupled receptors: PAC1-R, vasoactive intestinal peptidereceptor type 1 (“VPAC1-R”), and vasoactive intestinal peptide receptortype 2 (“VPAC2-R”). These receptors are expressed in diverse tissues.PAC1-R is particularly abundant in the nervous system (e.g., olfactorybulb, thalamus, hypothalamus, cerebellum, and spinal dorsal horn),pituitary, and adrenal glands. By contrast, VPAC1-R and VPAC2-R areexpressed mainly in the lung, liver, and testis, although they have beendetected in other tissues as well. VPAC1-R expression has been detectedin the nervous system (e.g., cerebral cortex and hippocampus), smoothmuscle cells of lung, liver, intestine, megakaryocytes, and platelets.VPAC1-R associates with receptor-associated membrane protein (“RAMP”,specifically RAIVIP2) (See, Christopoulos et al., J. Biol. Chem.,278:3293-3297, 2002). VPAC2-R expression profile includes the nervous(e.g., thalamus, hippocampus, brain stem, and dorsal root ganglia(“DRG”)), cardiovascular system, gastrointestinal system, pancreas, andreproductive systems (See, Usdin et al., Endocrin., 135:2662-2680, 1994;Sheward et al., Neurosci., 67:409-418, 1995).

PAC1-R is selective for PACAP38 and PACAP27. In particular, PAC1-R bindsto PACAP with 100-1000-fold greater affinity than VIP, i.e., K_(D)˜0.5nM for PACAP27/PACAP38 vs. K_(D)˜500 nM for VIP. Conversely, VPAC1-R andVPAC2-R have equal affinities for PACAP and VIP (K_(D)˜1 nM) (See,Schytz et al. 2010).

Upon activation, these receptors are all capable of causing downstreamproduction of cyclic adenosine monophosphate (“cAMP”), and/or activationof phospholipase C (“PLC”), and/or modulation of phospholipase D(“PLD”). In particular, PAC1-R is coupled to dual signal transductionpathways acting through cAMP and Ca²⁺, whereas VPAC1-R and VPAC2-R arecoupled principally to adenylyl cyclase. PAC1-R is coupled to G_(s)protein, which activates adenylyl cyclase to form cAMP that in turnactivates protein kinase A. PAC1-R also couples to Gq and therebyactivates PLC, which produces inositol phosphate, which increasescytosolic calcium release from intra-cellular calcium stores. There issome evidence for a role of PAC1-R in PLD activation (See, McCulloch etal., Ann. N. Y. Acad. Sci., 921:175-185, 2000). Another PACAP signalingpathway results in the elevation of intra-cellular sodium levels viaactivation of nonselective cation channels (See, Roy et al., AmericanJournal of Physiology: Regulatory, Integrative and ComparativePhysiology, 304(12):R1070-R1084, 2013).

PACAP is hypothesized to play a role in a multitude of diseases anddisorders, including but not limited to migraine, headache, and pain,though such a role for PACAP has not been clinically demonstrated.Migraines are believed to have a neurovascular component. Migrainesaffect approximately 10% of the adult population in the U.S. and aretypically accompanied by intense headaches. Approximately 20-30% ofmigraine sufferers experience aura, comprising focal neurologicalphenomena that precede and/or accompany the event. A role for PACAP inmigraine has been suggested by several observations: (1) plasma levelsof PACAP are elevated during migraine attacks (ictal), as compared tointerictal levels, in humans (see, Tuka et al., Cephalalgia,33(13):1085-1095 2013); (2) an infusion of PACAP38 triggered headachesin healthy subjects, and headaches followed by migraine-like attacks inmigraineurs (see, Schytz et al., Brain, 132:16-25, 2009; and Amin etal., Brain, 137:779-794, 2014, respectively); (3) PACAP-inducedvasodilation may play a role in neurogenic inflammation (see, Kaiser andRusso, Neuropeptides, 47:451-461, 2013); and (4) PACAP-induced migrainesare associated with photophobia, phonophobia, nausea, and respond totriptans (see, Amin et al., Brain, 32:140-149 2012). PACAP has also beenshown to induce vasodilation, photophobia, as well as mast celldegranulation and neuronal activation (See, Markovics et al.,Neurobiology of Disease, 45:633-644 2012; Baun et al., Cephalalgia,32(4):337-345 2012; Chan et al., Pharmacology & Therapeutics,129:332-351 2011).

One effective treatment for migraines is the administration of triptans,which are a family of tryptamine-based drugs, including sumatriptan andrizatriptan. Members of this family have an affinity for multipleserotonin receptors, including 5-HT_(1B), 5-HT_(1D), and 5-HT_(1F).Members of this family of drugs selectively constrict cerebral vessels,but also cause vasoconstrictive effects on coronary vessels (See,Durham, New Eng. J. Med., 350 (11):1073-75 2004). There is a theoreticalrisk of coronary spasm in patients with established heart diseasefollowing administration, and cardiac events after taking triptans inrare instances may occur. Accordingly, they are contraindicated for somepatients with coronary vascular disease.

Similarly, pain may often be addressed through the administration ofcertain narcotics or non-steroidal anti-inflammatory drugs (“NSAIDs”).However, the administration of these treatments often has negativeconsequences. NSAIDs have the potential to cause kidney failure,intestinal bleeding, and liver dysfunction. Narcotics have the potentialto cause nausea, vomiting, impaired mental functioning, and addiction.Therefore, it is desirable to identify alternative treatments for painin order to avoid certain of these negative consequences.

PACAP may also be involved in diseases and disorders other thanmigraine, headache, and pain. For example, PACAP may correlate to oreven play a causal role in anxiety disorders (WO 2012/106407);thrombocytopenia (WO 2004/062684); and inflammatory skin diseases (WO2010/007175). PACAP and PAC1-R polymorphisms are associated withpost-traumatic stress syndrome (“PTSD”) in females, major depressivedisorder, and generalized anxiety disorder, suggesting a role for PACAPin these conditions. Further, supporting a role for PACAP inthrombocytopenia, trisomy 18 patients have excess PACAP and exhibitdefective megakaryocyte maturation (See, Schytz et al. 2010; and Moodyet al., Curr. Opin. Endocrinol. Diabetes Obes., 18(1):61-67, 2011).

Also, PACAP and other neuropeptides, such as Calcitonin Gene-RelatedPeptide (“CGRP”), substance P, neurokinin A, bradykinin, andendothelin-1, are expressed in the lower urinary tract (“LUT”) (see,Arms and Vizzard, Handbook Exp. Pharmacol., 202:395-423, 2011) andreportedly may play a role in LUT dysfunction and urinary tractdisorders such as urinary tract infection (“UTI”), abnormal voiding,urinary urgency, nocturia, urinary incontinence, overactive bladder, andthe pain associated with such conditions.

PACAP and PACAP receptors have also been suggested to modulateinflammatory and neuropathic pain and have been implicated in bothpronociception and antinociception (See, Davis-Taber et al., J. Pain,9(5):449-56 2008). PACAP has also been reported to be required forspinal desensitization and the induction of neuropathic pain (See,Mabuchi et al., J. Neurosci., 24(33):7283-91, 2004). Additionally,morphine withdrawal behavior is reportedly modified in PACAP-receptordeficient mice further suggesting the role of PACAP in morphinewithdrawal anxiolytic response (See Martin et al., Mol. Brain Res.,110(1):109-18, 2003).

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention in general relates to anti-PACAPantibodies and antigen binding fragments thereof, preferably human,humanized, or chimerized anti-PACAP antibodies and antigen bindingfragments thereof, that antagonize, inhibit, neutralize, or block atleast one biological effect associated with human PACAP. In certainembodiments, the anti-PACAP antibodies and antigen binding fragmentsthereof inhibit or neutralize at least one biological effect elicited byPACAP, which includes PACAP27 and/or PACAP38, as discussed infra. Inother embodiments, the anti-PACAP antibodies and antigen bindingfragments thereof neutralize or inhibit PACAP activation of at least oneof PAC1-R, VPAC1-R, and/or VPAC2-R; neutralize or inhibit PACAPactivation of each of PAC1-R, VPAC1-R, and VPAC2-R; and/or neutralize orinhibit PACAP activation of PAC1-R; and/or inhibits PACAP binding to thecell surface, e.g., via a glycosaminoglycan (“GAG”). In yet otherembodiments, the anti-PACAP antibodies and antigen binding fragmentsthereof are capable of inhibiting PACAP binding to at least one ofPAC1-R, VPAC1-R, and/or VPAC2-R; are capable of inhibiting PACAP bindingto each of PAC1-R, VPAC1-R, and/or VPAC2-R; or are capable of inhibitingPACAP binding to PAC1-R. In other embodiments, the anti-PACAP antibodiesand antigen binding fragments thereof inhibit PACAP-induced cAMPproduction. In yet other embodiments, the anti-PACAP antibodies andantigen binding fragments thereof, alone or in combination, whenadministered to a subject, e.g., a human, reduce PACAP-inducedvasodilation, photophobia, mast cell degranulation, and/or neuronalactivation. In related embodiments, the human or humanized anti-PACAPantibodies and antigen binding fragments thereof are suitable fortreating a human subject having an acute, episodic or chronic conditionassociated with increased vasodilation, photophobia, mast celldegranulation, and/or neuronal activation.

In another embodiment, the method provides a eukaryotic host cell thatis mammalian selected from the group consisting of baby hamster kidney(“BHK”) cells; chinese hamster ovary (“CHO”) cells; mouse sertoli cells(“TM4” cells); African green monkey kidney cells (“VERO-76” cells);human cervical carcinoma (“HELA”) cells; canine kidney cells (“MDCK”);buffalo rat liver (“BRL”) cells; human lung cells; human liver (“HepG2”) cells; mouse mammary tumor (“MMT”) cells; TRI cells; MRC 5 cells;and FS4 cells. Preferably, the mammalian host cell is a CHO cell. Morepreferably, the mammalian host cell is a CHO K1 cell.

In a preferred embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof do not substantially interact with (bind) to VIP. Thepresent invention also encompasses the therapeutic use (as a monotherapyor combination therapy) and diagnostic use of such anti-PACAP antibodiesand antigen binding fragments thereof.

More particularly, anti-PACAP antibodies and antigen binding fragmentsthereof according to the invention can include human, humanized, andchimerized antibodies and fragments thereof, as well as scFvs,camelbodies, shark antibodies, nanobodies, Immunoglobulin New AntigenReceptor (“IgNAR”), fragment antigen binding (“Fab”) fragments, Fab′fragments, MetMab like antibodies, bispecific antibodies, monovalentantibody fragments, and F(ab′)₂ fragments. Additionally, anti-PACAPantibodies and antigen binding fragments thereof according to theinvention can substantially or entirely lack N-glycosylation and/orO-glycosylation. In one embodiment, the anti-PACAP antibodies andantigen binding fragments thereof comprise a human constant domain,e.g., that of IgG1, IgG2, IgG3, or IgG4 antibody or a fragment thereof.In another embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof may comprise an Fc region that has been modified toalter (enhance or impair) at least one of effector function, half-life,proteolysis, or glycosylation. For example, the Fc region may containone or more mutations that alters or eliminates N- and/orO-glycosylation.

In some embodiments, anti-PACAP antibodies and antigen binding fragmentsthereof bind to PACAP with a K_(D) of less than or equal to 5×10⁻⁵ M,10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M,10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M,5×10⁻¹³ M, or 10⁻¹³ M, e.g., as determined by ELISA, bio-layerinterferometry (“BLI”), Kinetic Exclusion Assay (KINEXA®, SapidyneInstruments, Boise, Id.), or SPR, e.g., at 25° or 37° C. Preferably, thehuman, humanized, or chimerized anti-PACAP antibodies and antigenbinding fragments thereof bind to PACAP with a K_(D) of less than orequal to 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, or 10⁻¹² M.Preferably, the human, humanized, or chimerized anti-PACAP antibodiesand antigen binding fragments thereof bind to PACAP with a K_(D) that isless than about 100 nM, less than about 40 nM, less than about 1 nM,less than about 100 pM, less than about 50 pM, or less than about 25 pM.Alternatively, the anti-PACAP antibodies and antigen binding fragmentsthereof bind to PACAP with a K_(D) that is between about 10 pM and about100 pM. In another embodiment, the human, humanized, or chimerizedanti-PACAP antibodies and antigen binding fragments thereof bind toPACAP with an off-rate (k_(off)) of less than or equal to 5×10⁻⁴ s⁻¹,10⁻⁴ s⁻¹, 5×10⁻⁵ s⁻¹, or 10⁻⁵ s⁻¹.

In yet another embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof will specifically bind to the linear or conformationalepitope(s) and/or compete for binding to the same linear orconformational epitope(s) on human PACAP as an anti-PACAP antibodyselected from the group consisting of Ab10 and Ab20 (the specific aminoacid sequences of the variable and constant regions of these anti-PACAPantibodies, and the nucleic acids that encode for such variable andconstant regions, and the epitopes bound thereby as determined usingalanine scanning methods are disclosed infra). In particular, theinvention embraces anti-PACAP antibodies and antigen binding fragmentsthereof that specifically bind to the same linear or conformationalepitope(s) on human PACAP as an anti-PACAP antibody selected from thegroup consisting of Ab10 and Ab20. As disclosed infra, in exemplaryembodiments, the epitope(s) are determined using alanine scanningmutation strategy.

In yet another embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof include human, humanized or chimerized anti-PACAPantibodies or antibody fragments which bind to the identical epitopes asany one of Ab10 or Ab20 or a binding fragment of any one of theforegoing.

In yet another embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof include human, humanized or chimerized anti-PACAPantibodies or antibody fragments that specifically bind to an epitope onhuman PACAP or a fragment or variant thereof containing thecorresponding amino acid residues wherein said epitope is selected fromthe group consisting of:

a. at least one of residues 19, 22, 23 and 27 of human PACAP;

b. at least one of residues 19, 22, 23, 24 and 27 of human PACAP;

c. at least two of the residues of any one of (a)-(b);

d. at least three of the residues of any one of (a)-(b);

e. at least four of the residues of any one of (a)-(b);

f. all five of the residues of (b).

In yet another embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof include human, humanized or chimerized anti-PACAPantibodies or antibody fragments that specifically bind to an epitope onhuman PACAP, or a fragment or variant thereof containing thecorresponding amino acid residues that includes one or more of residues19, 22, 23 and 27 of human PACAP.

In yet another embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof include human, humanized or chimerized anti-PACAPantibodies or antibody fragments that specifically bind to an epitope onhuman PACAP or a fragment or variant thereof containing thecorresponding amino acid residues that is present in human wild-typePACAP38 but not human wild-type human PACAP27.

In yet another embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof include human, humanized or chimerized anti-PACAPantibodies or antibody fragments that specifically bind to an whichspecifically binds to an epitope on human PACAP or a fragment or variantthereof containing the corresponding amino acid residues, wherein saidepitope is identified by alanine scanning, e.g., as disclosed in Example12 or another art-recognized method.

In yet another embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof include human, humanized or chimerized anti-PACAPantibodies or antibody fragments that specifically bind to an epitope onhuman PACAP or a fragment or variant thereof containing thecorresponding amino acid residues, wherein said epitope consists of theresidues of (a) or (b) above.

In yet another embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof include human, humanized or chimerized anti-PACAPantibodies or antibody fragments which specifically binds to an epitopeon human PACAP or a fragment or variant thereof containing thecorresponding amino acid residues that is present in human wild-typePACAP38 and in human wild-type human PACAP27.

In yet another embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof include human, humanized or chimerized anti-PACAPantibodies or antibody fragments that specifically bind to humanwild-type human PACAP38 but which does not bind or appreciably bind tohuman wild-type human PACAP27.

In yet another embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof include human, humanized or chimerized anti-PACAPantibodies or antibody fragments which has a K_(D) for human PACAP38which is at least 10 fold, 100 fold, 1,000 fold, 10,000 fold, or 100,000fold lower (stronger) than the K_(D) of said antibody or antibodyfragment to human PACAP27.

In yet another embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof include human, humanized or chimerized anti-PACAPantibodies or antibody fragments which do not bind to or does notappreciably bind to human Vasoactive Intestinal Peptide (“VIP”).

In yet another embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof include human, humanized or chimerized anti-PACAPantibodies or antibody fragments which have a K_(D) for human PACAPwhich is at least 10, 100, 1,000, 10,000 or 100,000 fold less (weaker)than the K_(D) of said antibody or antibody fragment to human VIP.

In some embodiments, the present invention provides an anti-PACAPantibodies and antigen binding fragments thereof, are preferably human,humanized, or chimerized anti-PACAP antibodies and antigen bindingfragments thereof, comprising at least 2 complementarity determiningregions (“CDRs”), or at least 3 CDRs, or at least 4 CDRs, or at least 5CDRs, or all six CDRs of an anti-PACAP antibody selected from the groupconsisting of Ab10 and Ab20. In instances where all 6 CDRs are notpresent, preferably at least the V_(H) CDR3 and V_(L) CDR3 are present.In exemplary embodiments, the antibodies and antigen binding fragmentsthereof comprise the variable heavy (“V_(H)”) chain and/or the variablelight (“V_(L)”) chain of one of Ab10 or Ab20.

In a specific embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof according to the invention, are human, humanized, orchimerized anti-PACAP antibodies or antigen binding fragments thereof,and comprise (a) a variable heavy chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 404; a CDR2 sequence consisting of SEQ ID NO:406; and a CDR3 sequence consisting of SEQ ID NO: 408; and/or (b) avariable light chain comprising a CDR1 sequence consisting of SEQ ID NO:424; a CDR2 sequence consisting of SEQ ID NO: 426; and a CDR3 sequenceconsisting of SEQ ID NO: 428. Alternatively, the anti-PACAP antibodiesand antigen binding fragments thereof can comprise (a) a variable heavychain comprising an amino acid sequence with at least 80, 85, 90, 95,96, 97, 98, or 99% sequence identity to SEQ ID NO: 402, and/or (b) avariable light chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 422. Inanother embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof comprise (a) a variable heavy chain having the aminoacid sequence of SEQ ID NO: 402, and/or (b) a variable light chainhaving the amino acid sequence of SEQ ID NO: 422. More specifically, theanti-PACAP antibodies and antigen binding fragments thereof can comprise(a) a heavy chain having the amino acid sequence of SEQ ID NO: 401,and/or (b) a light chain having the amino acid sequence of SEQ ID NO:421.

In another specific embodiment, the anti-PACAP antibodies and antigenbinding fragments thereof according to the invention, are human,humanized, or chimerized anti-PACAP antibodies or antigen bindingfragments thereof, and comprise (a) a variable heavy chain comprising aCDR1 sequence consisting of SEQ ID NO: 444; a CDR2 sequence consistingof SEQ ID NO: 446; and a CDR3 sequence consisting of SEQ ID NO: 448;and/or (b) a variable light chain comprising a CDR1 sequence consistingof SEQ ID NO: 464; a CDR2 sequence consisting of SEQ ID NO: 466; and aCDR3 sequence consisting of SEQ ID NO: 468. Alternatively, theanti-PACAP antibodies and antigen binding fragments thereof can comprise(a) a variable heavy chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:442, and/or (b) a variable light chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO: 462. In another embodiment, the anti-PACAP antibodies andantigen binding fragments thereof and comprise (a) a variable heavychain having the amino acid sequence of SEQ ID NO: 442, and/or (b) avariable light chain having the amino acid sequence of SEQ ID NO: 462.More specifically, the anti-PACAP antibodies and antigen bindingfragments thereof can comprise (a) a heavy chain having the amino acidsequence of SEQ ID NO: 441, and/or (b) a light chain having the aminoacid sequence of SEQ ID NO: 461.

Also, in some embodiments the anti-PACAP antibodies and antigen bindingfragments may comprise sequence variants of any of the disclosedantibodies which are modified by mutagenesis, e.g., affinity maturationto alter one or more properties such as binding affinity orimmunogenicity.

In another embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof are directly or indirectly attached to another moiety,such as a detectable label or therapeutic agent.

In another embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof inhibit or neutralize at least one biological effectelicited by PACAP; neutralize or inhibit PACAP activation of at leastone of PAC1-R, VPAC1-R, and/or VPAC2-R; neutralize or inhibit PACAPactivation of each of PAC1-R, VPAC1-R, and VPAC2-R; neutralize orinhibit PACAP activation of PAC1-R; are capable of inhibiting PACAPbinding to at least one of PAC1-R, VPAC1-R, and/or VPAC2-R; are capableof inhibiting PACAP binding to each of PAC1-R, VPAC1-R, and/or VPAC2-R;are capable of inhibiting PACAP binding to PAC1-R; and/or inhibits PACAPbinding to the cell surface, e.g., via a GAG; inhibit PACAP-induced cAMPproduction; and/or when administered to a subject reduce PACAP-inducedvasodilation, photophobia, mast cell degranulation, and/or neuronalactivation.

In another embodiment, the human, or humanized, anti-PACAP antibodiesand antigen binding fragments thereof are suitable for treating a humansubject having an acute, episodic, or chronic condition associated withincreased vasodilation, photophobia, mast cell degranulation, and/orneuronal activation.

In another embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof do not substantially interact with (i.e., bind to)VIP. Preferably, the anti-PACAP antibodies and antigen binding fragmentsthereof have stronger affinity for PACAP as compared to VIP, i.e.,although there is some cross-reactivity, the antibodies preferentiallybind to PACAP as compared to VIP. For example, the affinity of saidantibodies and antigen binding fragments thereof to PACAP is at least10-fold, 30-fold, 100-fold, 300-fold, 1000-fold, 3000-fold, 10000-fold,30000-fold, 100000-fold, 300000-fold, 1000000-fold, 3000000-fold,10000000-fold, 30000000-fold, or stronger than the affinity of saidantibodies and antigen binding fragments thereof to VIP (e.g., the KD ofsaid antibody or fragment for binding to human PACAP is 10-fold,30-fold, 100-fold, 300-fold, 1000-fold, 3000-fold, 10000-fold,30000-fold, 100000-fold, 300000-fold, 1000000-fold, 3000000-fold,10000000-fold, or 30000000-fold lower than the K_(D) for binding toVIP).

In one embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof are attached to at least one effector moiety, e.g.,which comprises a chemical linker. In another embodiment, the anti-PACAPantibodies and antigen binding fragments thereof are attached to one ormore detectable moieties, e.g., which comprise a fluorescent dye,enzyme, substrate, bioluminescent material, radioactive material,chemiluminescent moiety, or mixtures thereof.

In one embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof are attached to one or more functional moieties.

The invention also contemplates antibodies, e.g., anti-idiotypicantibodies, produced against an anti-PACAP antibodies and antigenbinding fragments thereof as described above. Furthermore, the inventionprovides a method of using the anti-idiotypic antibody to monitor the invivo levels of said anti-PACAP antibodies and antigen binding fragmentsthereof in a subject or to neutralize said anti-PACAP antibody in asubject being administered said anti-PACAP antibody or antigen bindingfragment thereof.

Moreover, the present invention encompasses a composition suitable fortherapeutic, prophylactic, or a diagnostic use comprising atherapeutically, prophylactically, or diagnostically effective amount ofat least one anti-PACAP antibody or antigen binding fragment asdescribed herein. In particular, compositions and dosage formscontaining the subject anti-PACAP antibodies or binding fragmentsthereof for use in treating or preventing migraine or other headacheindications are provided herein. Also provided herein are dosage formscontaining the subject anti-PACAP antibodies or binding fragmentsthereof for use in treating or preventing photophobia. The compositionmay be suitable for subcutaneous administration, intra-muscularadministration, and/or intravenous administration. The composition maybe lyophilized. In some embodiments, the composition further comprises apharmaceutically acceptable diluent, carrier, solubilizer, emulsifier,preservative, or mixture thereof.

Additionally, in some embodiments, the composition further comprisesanother active agent, e.g., a chemotherapeutic, an analgesic, ananti-inflammatory, an immunosuppressant, a cytokine, anantiproliferative, and an antiemetic. Preferably, the other therapeuticagent is an analgesic, e.g., an NSAID, an opioid analgesic, an antibody(e.g., an anti-human Nerve Growth Factor (“NGF”) antibody or antibodyfragment; or an anti-human CGRP or anti-human CGRP-receptor antibody orantibody fragment); or a non-antibody biologic, such as an NGF or CGRPpolypeptide fragment or conjugate; or BOTOX® (Botulinum toxin). SuitableNSAIDs for use in combination with the subject anti-PACAP antibodiesinclude, but are not limited to, a cyclooxygenase 1 and/orcyclooxygenase 2 inhibitor; propionic acid derivatives includingibuprofen, naproxen, naprosyn, diclofenac, and ketoprofen; acetic acidderivatives including tolmetin and sulindac; fenamic acid derivativesincluding mefenamic acid and meclofenamic acid; biphenylcarboxylic acidderivatives including diflunisal and flufenisal; and oxicams includingpiroxim, sudoxicam, and isoxicam. Suitable opioid analgesics for use incombination with the subject anti-PACAP antibodies include, e.g.,codeine, dihydrocodeine, morphine or a morphine derivative orpharmaceutically acceptable salt thereof, diacetylmorphine, hydrocodone,hydromorphone, levorphanol, oxymorphone, alfentanil, buprenorphine,butorphanol, fentanyl, sufentanil, meperidine, methadone, nalbuphine,propoxyphene, and pentazocine, or pharmaceutically acceptable saltsthereof. The combined administration of the opioid analgesic and theanti-PACAP antibody or antigen binding fragment thereof may increase theanalgesic effect elicited thereby.

The present invention further contemplates an isolated nucleic acidsequence or nucleic acid sequences encoding an anti-PACAP antibody orantigen binding fragment described herein, as well as a vector orvectors containing these isolated nucleic acid sequence or sequences.

Additionally, the invention provides a host cell comprising theseisolated nucleic acid sequence or sequences or the vector or set forthabove. The host cell may be a eukaryotic host cell that is mammalian,selected from the group consisting of baby hamster kidney (“BHK”) cells;chinese hamster ovary (“CHO”) cells; mouse sertoli cells (“TM4” cells);African green monkey kidney cells (“VERO-76” cells); human cervicalcarcinoma (“HELA”) cells; canine kidney cells (“MDCK”); buffalo ratliver (“BRL”) cells; human lung cells; human liver (“Hep G2”) cells;mouse mammary tumor (“MMT”) cells; TRI cells; MRC 5 cells; and FS4cells. Preferably, the mammalian host cell is a CHO cell. Morepreferably, the mammalian host cell is a CHO K1 cell. The host cell maybe a prokaryotic cell, i.e., bacterial cell, or a eukaryotic cell,including a mammalian, fungal, yeast, avian, or insect cell. In oneembodiment, the host cell is a filamentous fungus or is a yeast cell.Preferably, the yeast species is of the genus Pichia. Most preferably,the species of Pichia is selected from Pichia pastoris, Pichiamethanolica, and Hansenula polymorpha (Pichia angusta).

The invention further provides a method of expressing anti-PACAPantibodies and antigen binding fragments thereof, typically human,humanized, or chimeric antibodies and antigen binding fragments thereof,the method comprising culturing the host cell described herein underconditions that provide for expression of said antibody or antigenbinding fragment thereof. The host cell may be a cell culture, such as aChinese hamster ovary (“CHO”) cell or a polyploid yeast culture thatstably expresses and secretes into the culture medium at least 10-25mg/liter of said antibody or antigen binding fragment thereof. Thepolyploid yeast may be made by a method that comprises: (i) introducingat least one expression vector containing one or more heterologouspolynucleotides encoding said antibody operably linked to a promoter anda signal sequence into a haploid yeast cell; (ii) producing by mating orspheroplast fusion a polyploid yeast from said first and/or secondhaploid yeast cell; (iii) selecting polyploid yeast cells that stablyexpress said antibody; and (iv) producing stable polyploid yeastcultures from said polyploid yeast cells that stably express saidantibody into the culture medium. Preferably, the yeast species is ofthe genus Pichia.

In other embodiments, the mammalian cell culture may be made by a methodthat comprises: (i) introducing at least one expression vectorcontaining one or more heterologous polynucleotides encoding saidantibody operably linked to a promoter and a signal sequence into amammalian cell; (ii) producing single cells for culturing to express oneor more heterologous polynucleotides encoding said antibody; (iii)selecting a mammalian cell that stably expresses said antibody; and (iv)producing cell cultures from said mammalian cell that stably expressessaid antibody into the culture medium. Preferably, the mammalian speciesare CHO cells.

The invention further relates to the therapeutic and diagnostic uses ofanti-PACAP antibodies and antigen binding fragments thereof, preferablya human antibody, humanized antibody, or chimeric antibody, or afragment thereof.

In one embodiment, the invention provides a method for blocking,inhibiting, or neutralizing one or more biological effects associatedwith PACAP in a subject comprising administering to a subject aneffective amount of a human or humanized or chimerized anti-PACAPantibody or antigen binding fragment thereof that antagonizes, inhibits,neutralizes, or blocks at least one biological effect associated withhuman PACAP. In a specific embodiment, the method employs an anti-PACAPantibody or antigen binding fragment thereof that specifically binds tothe same or overlapping linear or conformational epitope(s) and/orcompetes for binding to the same or overlapping linear or conformationalepitope(s) on human PACAP as an anti-PACAP antibody selected from Ab10or Ab20.

In another embodiment, the invention provides a method for blocking,inhibiting, or neutralizing one or more biological effects associatedwith PACAP in a subject comprising administering to a subject aneffective amount of a human, humanized, or chimerized anti-PACAPantibody or antigen binding fragment thereof that antagonizes, inhibits,neutralizes, or blocks at least one biological effect associated withhuman PACAP and that does not substantially interact with (bind) VIP,e.g., the anti-PACAP antibody or antigen binding fragment thereof hasstronger affinity for PACAP as compared to VIP, i.e., although there issome cross-reactivity, the antibodies preferentially bind to PACAP ascompared to VIP. For example, the affinity of said antibody or antigenbinding fragment thereof to PACAP is at least 10-fold, 30-fold,100-fold, 300-fold, 1000-fold, 3000-fold, 10000-fold, 30000-fold,100000-fold, 300000-fold, 1000000-fold, 3000000-fold, 10000000-fold,30000000-fold, or higher than the affinity of said antibody or antigenbinding fragment thereof to VIP (e.g., the KD of said antibody orfragment for binding to human PACAP is 10-fold, 30-fold, 100-fold,300-fold, 1000-fold, 3000-fold, 10000-fold, 30000-fold, 100000-fold,300000-fold, 1000000-fold, 3000000-fold, 10000000-fold, 30000000-fold,or lower than the K_(D) for binding to VIP). In a specific embodiment,the method employs an anti-PACAP antibody or antigen binding fragmentthereof that specifically binds to the same or overlapping linear orconformational epitope(s) and/or competes for binding to the same oroverlapping linear or conformational epitope(s) on human PACAP as ananti-PACAP antibody selected from Ab10 or Ab20.

In yet another embodiment, the invention provides a method for blocking,inhibiting, or neutralizing one or more biological effects associatedwith PACAP in a subject comprising administering to a subject aneffective amount of a human, humanized, or chimerized anti-PACAPantibody or antigen binding fragment thereof that inhibits orneutralizes at least one biological effect elicited by PACAP;neutralizes or inhibits PACAP activation of at least one of PAC1-R,VPAC1-R, and/or VPAC2-R; neutralizes or inhibits PACAP activation ofeach of PAC1-R, VPAC1-R, and VPAC2-R; neutralizes or inhibits PACAPactivation of PAC1-R; is capable of inhibiting PACAP binding to at leastone of PAC1-R, VPAC1-R, and/or VPAC2-R; is capable of inhibiting PACAPbinding to each of PAC1-R, VPAC1-R, and/or VPAC2-R; is capable ofinhibiting PACAP binding to PAC1-R; and/or is capable of inhibitingPACAP binding to the cell surface, e.g., via GAG; inhibits PACAP-inducedcAMP production; and/or when administered to a subject reducesPACAP-induced vasodilation, photophobia, mast cell degranulation, and/orneuronal activation. In a specific embodiment, the method employs ananti-PACAP antibody or antigen binding fragment thereof thatspecifically binds to the same or overlapping linear or conformationalepitope(s) and/or competes for binding to the same or overlapping linearor conformational epitope(s) on human PACAP as an anti-PACAP antibodyselected from Ab10 or Ab20.

In another embodiment, the invention provides a method for treating orpreventing the onset, frequency, severity, or duration of headache ormigraine in a subject comprising administering to a subject an effectiveamount of a human, humanized, or chimerized anti-PACAP antibody orantigen binding fragment thereof that inhibits or neutralizes at leastone biological effect elicited by PACAP; neutralizes or inhibits PACAPactivation of at least one of PAC1-R, VPAC1-R, and/or VPAC2-R;neutralizes or inhibits PACAP activation of each of PAC1-R, VPAC1-R, andVPAC2-R; neutralizes or inhibits PACAP activation of PAC1-R; is capableof inhibiting PACAP binding to at least one of PAC1-R, VPAC1-R, and/orVPAC2-R; is capable of inhibiting PACAP binding to each of PAC1-R,VPAC1-R, and/or VPAC2-R; is capable of inhibiting PACAP binding toPAC1-R; and/or is capable of inhibiting PACAP binding to the cellsurface, e.g., via GAG; inhibits PACAP-induced cAMP production; and/orwhen administered to a subject reduces PACAP-induced vasodilation,photophobia, mast cell degranulation, and/or neuronal activation. Inanother embodiment, the invention provides a method for treating orpreventing in a human subject an acute, episodic, or chronic conditionassociated with increased vasodilation, photophobia, mast celldegranulation, and/or neuronal activation.

In a specific embodiment, the method employs an anti-PACAP antibody orantigen binding fragment thereof that specifically binds to the same oroverlapping linear or conformational epitope(s) and/or competes forbinding to the same or overlapping linear or conformational epitope(s)on human PACAP as an anti-PACAP antibody selected from Ab10 or Ab20. Theepitope can be identified using an alanine scanning mutation strategy,for example.

In a specific embodiment, the headache or migraine treated and/orprevented by administration of the subject anti-PACAP antibodies andantigen binding fragments thereof is selected from migraine with orwithout aura, hemiplegic migraine, cluster headache, migrainousneuralgia, chronic headache, and tension headache.

In another specific embodiment, the subject has a ocular disorderassociated with photophobia selected from the group consisting ofachromatopsia, aniridia, photophobia caused by an anticholinergic drug,aphakia (absence of the lens of the eye), buphthalmos (abnormally narrowangle between the cornea and iris), cataracts, cone dystrophy,congenital abnormalities of the eye, viral conjunctivitis (“pink eye”),corneal abrasion, corneal dystrophy, corneal ulcer, disruption of thecorneal epithelium, ectopia lentis, endophthalmitis, eye trauma causedby disease, injury, or infection such as chalazion, episcleritis,glaucoma, keratoconus, or optic nerve hypoplasia, hydrophthalmos, orcongenital glaucoma iritis, optic neuritis, pigment dispersion syndrome,pupillary dilation (naturally or chemically induced), retinaldetachment, scarring of the cornea or sclera, and uveitis.

In another specific embodiment, the subject has a nervous system-relatedor neurological condition associated with photophobia selected from thegroup consisting of autism spectrum disorders, chiari malformation,dyslexia, encephalitis including myalgic encephalomyelitis (also knownas “chronic fatigue syndrome”), meningitis, subarachnoid hemorrhage,tumor of the posterior cranial fossa, ankylosing spondylitis, albinism,ariboflavinosis, benzodiazepines (long term use of or withdrawal frombenzodiazepines), chemotherapy, chikungunya, cystinosis, Ehlers-Danlossyndrome, hangover, influenza, infectious mononucleosis, magnesiumdeficiency, mercury poisoning, migraine, rabies, and tyrosinemia type II(also known as “Richner-Hanhart syndrome”).

In another specific embodiment, the subject has a photophobia-associateddisorder selected from the group consisting of migraine (with or withoutaura), iritis, uveitis, meningitis, depression, bipolar disorder,cluster headache or anther trigeminal autonomic cephalalgia (“TAC”) orblepharospasm, depression, agoraphobia, Post-Traumatic Stress Disorder(“PTSD”), traumatic brain injury, and bipolar disorder.

In another embodiment, the invention provides a method for neutralizingPACAP-induced PAC1-R, VPAC1-R, and/or VPAC2-R signaling, comprisingadministering to a subject in need thereof an effective amount of ananti-PACAP antibody or antigen binding fragment thereof thatspecifically binds to the same or overlapping linear or conformationalepitope(s) and/or competes for binding to the same or overlapping linearor conformational epitope(s) on human PACAP as an anti-PACAP antibodyselected from Ab10 or Ab20.

In another embodiment, the invention provides a method for inhibitingPACAP-induced cAMP production, comprising administering to a subject inneed thereof an effective amount of an anti-PACAP antibody or antigenbinding fragment thereof that specifically binds to the same oroverlapping linear or conformational epitope(s) and/or competes forbinding to the same or overlapping linear or conformational epitope(s)on human PACAP as an anti-PACAP antibody selected from Ab10 or Ab20.

In yet another embodiment, the invention provides a method forinhibiting PACAP-induced vasodilation, photophobia, mast celldegranulation, and/or neuronal activation, comprising administering to asubject in need thereof an effective amount of an anti-PACAP antibody orantigen binding fragment thereof that specifically binds to the same oroverlapping linear or conformational epitope(s) and/or competes forbinding to the same or overlapping linear or conformational epitope(s)on human PACAP as an anti-PACAP antibody selected from Ab10 or Ab20.

In yet another embodiment, the invention provides a method for treatingor preventing a condition associated with elevated PACAP levels in asubject, comprising administering to a subject in need thereof aneffective amount of an anti-PACAP antibody or antigen binding fragmentthereof that specifically binds to the same or overlapping linear orconformational epitope(s) and/or competes for binding to the same oroverlapping linear or conformational epitope(s) on human PACAP as ananti-PACAP antibody selected from Ab10 or Ab20. The epitope can beidentified using an alanine scanning mutation strategy, for example.

Exemplary anti-PACAP antibodies and antigen binding fragments thereofsuitable for use in this invention comprise a V_(H) chain having anamino acid sequence with at least 80, 85, 90, 95, 96, 97, 98, 99 or 100%sequence identity to a V_(H) chain selected from SEQ ID NO: 402 and 442,and/or a V_(L) chain having an amino acid sequence with at least 80, 85,90, 95, 96, 97, 98, 99, or 100% sequence identity to a V_(L) chainselected from selected from SEQ ID NO: 422 and 462, and/or at least 2,3, 4, 5, or all 6 CDRs comprised therein.

In one embodiment, the anti-PACAP antibody or antigen binding fragmentthereof employed in the methods binds to PACAP27 and/or PACAP38 andblocks PACAP27 and/or PACAP38 binding to PAC1-R, VPAC1-R, and/orVPAC2-R. In another embodiment, the anti-PACAP antibody or antigenbinding fragment thereof employed in the methods binds to PACAP27 and/orPACAP38 and blocks PACAP27 and/or PACAP38 binding to each of PAC1-R,VPAC1-R, and VPAC2-R. Preferably, the anti-PACAP antibody or antigenbinding fragment thereof binds to PACAP27 and/or PACAP38 and blocksPACAP27 and/or PACAP38 binding to PAC1-R.

More particularly, anti-PACAP antibodies and antigen binding fragmentsthereof employed in the methods according to the invention may includehuman, humanized, and chimerized antibodies and fragments thereof, aswell as scFvs, camelbodies, shark antibodies, nanobodies, IgNAR, Fabfragments, Fab′ fragments, MetMab like antibodies, bispecificantibodies, monovalent antibody fragments, and F(ab′)₂ fragments.Additionally, the anti-PACAP antibody or antigen binding fragmentthereof employed by the methods according to the invention maysubstantially or entirely lack N-glycosylation and/or O-glycosylation.In one embodiment, the anti-PACAP antibody or antigen binding fragmentthereof used in the encompassed methods comprises a human constantdomain, e.g., an IgG1, IgG2, IgG3, or IgG4 antibody. In anotherembodiment, the anti-PACAP antibody or antigen binding fragment thereofcomprises an Fc region that has been modified to alter (enhance orimpair) at least one of effector function, half-life, proteolysis, orglycosylation. For example, the Fc region may contain one or moremutations that alters or eliminates N- and/or O-glycosylation.

In one embodiment, the subject methods employ an anti-PACAP antibody orantigen binding fragment thereof that binds to PACAP with a K_(D) ofless than or equal to 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M,10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, or 10⁻¹³ M. Preferably, thehuman, humanized, or chimerized anti-PACAP antibody or antigen bindingfragment thereof binds to PACAP with a K_(D) of less than or equal to5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, or 10⁻¹² M. Morepreferably, the methods employ a human, humanized, or chimerizedanti-PACAP antibody or antigen binding fragment thereof that binds toPACAP with a K_(D) that is less than about 100 nM, less than about 40nM, less than about 1 nM, less than about 100 pM, less than about 50 pM,or less than about 25 pM. Alternatively, the anti-PACAP antibody orantigen binding fragment thereof binds to PACAP with a K_(D) that isbetween about 10 pM and about 100 pM. In another embodiment, the human,humanized or chimerized anti-PACAP antibody or antigen binding fragmentthereof binds to PACAP with an off-rate (k_(off)) of less than or equalto 5×10⁻⁴ s⁻¹, 10⁴ s⁻¹, 5×10⁻⁵ s⁻¹, or 10⁻⁵ s⁻¹.

In another embodiment, the anti-PACAP antibody or antigen bindingfragment thereof used in the subject methods is directly or indirectlyattached to another moiety, such as a detectable label or therapeuticagent; is attached to at least one effector moiety, e.g., whichcomprises a chemical linker; and/or is attached to one or moredetectable moieties, e.g., which comprises a fluorescent dye, enzyme,substrate, bioluminescent material, radioactive material,chemiluminescent moiety, or mixtures thereof; and/or is attached to oneor more functional moieties.

In another embodiment, the method further comprises administeringseparately or co-administering another agent, e.g., selected from achemotherapeutic, an analgesic, an anti-inflammatory, animmunosuppressant, a cytokine, an antiproliferative, and an antiemetic.Preferably, the other therapeutic agent is an analgesic, e.g., an NSAID(such as a cyclooxygenase 1 and/or cyclooxygenase 2 inhibitor; propionicacid derivatives including ibuprofen, naproxen, naprosyn, diclofenac,and ketoprofen; acetic acid derivatives including tolmetin and sulindac;fenamic acid derivatives including mefenamic acid and meclofenamic acid;biphenylcarboxylic acid derivatives including diflunisal and flufenisal;and oxicams including piroxim, sudoxicam, and isoxicam), an opioidanalgesic (such as morphine or a morphine derivative or pharmaceuticallyacceptable salt thereof; codeine, dihydrocodeine, diacetylmorphine,hydrocodone, hydromorphone, levorphanol, oxymorphone, alfentanil,buprenorphine, butorphanol, fentanyl, sufentanil, meperidine, methadone,nalbuphine, propoxyphene and pentazocine or pharmaceutically acceptablesalts thereof), another antibody (such as an anti-NGF antibody orantibody fragment or an anti-CGRP or anti-CGRP receptor (“anti-CGRP-R”)antibody or antibody fragment), or a non-antibody biologic, e.g.,BOTOX®.

In one embodiment, the combined administration of the opioid analgesicand the anti-PACAP antibody or antigen binding fragment thereof increasethe analgesic effect as compared to either the opioid analgesic or theanti-PACAP antibody or antigen binding fragment thereof administeredalone.

In another embodiment, the subject has previously been treated (“atreated subject”) and received an anti-CGRP or anti-CGRP-R antibody orantibody fragment thereof. The treated subject may be a migraineur whodid not adequately respond to anti-CGRP or anti-CGRP-R antibodytreatment (“poor responder”). Alternatively, the treated subject mayhave previously received at least one anti-CGRP or anti-CGRP-R antibodyor antibody fragment thereof administration, and has elicited an immuneresponse to said antibody or antibody fragment thereof. Exemplaryanti-CGRP and anti-CGRP-R antibodies and antibody fragments thereof aredisclosed in U.S. Pat. Nos. 9,102,731; 9,115,194; 8,734,802; 8,623,366;8,597,649; and 8,586,045; and U.S. Patent Application Publication Nos.20120294822, 20120294802, and 20120294797, the contents of each whichare incorporated by reference in their entireties herein.

An aspect of the present invention in general relates to anti-PACAPantibodies and antigen binding fragments thereof, preferably human,humanized, or chimerized anti-human PACAP antibodies or antibodyfragments thereof that may antagonize, inhibit, neutralize or block atleast one biological effect associated with human PACAP.

Moreover, the invention pertains to anti-PACAP antibodies and antigenbinding fragments thereof that may include human, humanized orchimerized anti-PACAP antibodies or antibody fragments thereof thatspecifically compete for binding to human PACAP with an antibody thatmay be selected from the group consisting of Ab10 and Ab20, or anantigen-binding fragment thereof.

Additionally, the anti-PACAP antibodies and antigen binding fragments ofthe invention may include human, humanized or chimerized anti-PACAPantibodies or antibody fragments that may specifically bind to at leastone linear or conformational epitope bound by an anti-PACAP antibodythat may be selected from the group consisting of Ab10 and Ab20, or anantigen-binding fragment thereof. The epitope may be identified byalanine scanning, e.g., as disclosed in Example 12 or anotherart-recognized method.

Also, in another embodiment, the anti-PACAP antibodies and antigenbinding fragments thereof of the invention may include human, humanizedor chimerized anti-PACAP antibodies or antibody fragments thereof whichbind to the identical epitopes as any one of Ab10 or Ab20, or anantigen-binding fragment thereof. The epitope may be identified byalanine scanning, e.g., as disclosed in Example 12 or anotherart-recognized method.

In a further embodiment of the invention, the anti-PACAP antibodies orantigen binding fragments thereof of the invention may include human,humanized or chimerized anti-PACAP antibodies or antibody fragmentswhich may specifically bind to an epitope on human PACAP or a fragmentor variant thereof containing the corresponding amino acid residueswherein said epitope includes one or more of the following:

(i) at least one of residues 19, 22, 23 and 27 of human PACAP;(ii) at least one of residues 19, 22, 23, 24 and 27 of human PACAP;(iii) at least two of the residues of any one of (i)-(ii);(iv) at least three of the residues of any one of (i)-(ii);(v) at least four of the residues of any one of (i)-(ii); and(v) all five of the residues of (ii).

In a specific embodiment of the invention, anti-PACAP antibodies orantigen binding fragments thereof of the invention may include human,humanized or chimerized anti-PACAP antibodies or antibody fragmentswhich specifically bind to an epitope on human PACAP, or a fragment orvariant thereof that may contain the corresponding amino acid residuesthat may include residues 19, 22, 23 and 27 of human PACAP. Also, inanother embodiment of the invention, the anti-PACAP antibodies orantigen binding fragments thereof may include human, humanized orchimerized anti-PACAP antibodies or antibody fragments thereof, whichspecifically bind to an epitope on human PACAP (or a fragment or variantthereof that may contain the corresponding amino acid residues that maybe present in human wild-type PACAP38) but not human wild-type humanPACAP27. The epitope may be identified by alanine scanning, e.g., asdisclosed in Example 12 or another art-recognized method.

In an additional embodiment of the invention, anti-PACAP antibodies orantigen binding fragments thereof of the invention may include human,humanized or chimerized anti-PACAP antibodies or antibody fragmentsthereof which may specifically bind to an epitope on human PACAP or afragment or variant thereof that contain the corresponding amino acidresidues, wherein said epitope may consist of the residues of (i)-(ii)as described above. The epitope may be identified by alanine scanning,e.g., as disclosed in Example 12 or another art-recognized method.

Another aspect of the invention also embraces anti-PACAP antibodies orantigen binding fragments thereof that may include human, humanized orchimerized anti-PACAP antibodies or antibody fragment which specificallybind to an epitope on human PACAP (or a fragment or variant thereofcontaining the corresponding amino acid residues) that may be present inhuman wild-type PACAP38 and in human wild-type human PACAP27.

Additionally, the anti-PACAP antibodies or antigen binding fragmentsthereof may include human, humanized or chimerized anti-PACAP antibodiesor antibody fragments thereof, which may specifically bind to humanwild-type human PACAP38 but which may not bind or appreciably bind tohuman wild-type human PACAP27. In another embodiment of the invention,the anti-PACAP antibodies and antigen binding fragments thereof of theinvention may include human, humanized or chimerized anti-PACAPantibodies or antibody fragments thereof which may have a K_(D) forhuman PACAP38 which may be at least 10, 100, 1000, 10,000 or 100,000fold lower (stronger) than the K_(D) of said antibody or antibodyfragment to human PACAP27.

In another embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof of the invention may include human, humanized orchimerized anti-PACAP antibodies or antibody fragments thereof which donot bind to or do not appreciably bind to human Vasoactive IntestinalPeptide (“VIP”). Said anti-PACAP antibodies or antibody fragmentsthereof may have a K_(D) for human PACAP which may be at least 10, 100,1000, 10,000 or 100,000 fold lower (stronger) than the K_(D) of saidantibody or antibody fragment to human VIP.

In embodiments of the invention, the human, humanized or chimerizedanti-PACAP antibody or antibody fragment as disclosed herein, mayinhibit or neutralize at least one biological effect elicited by humanPACAP.

In another embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof of the invention may include human, humanized orchimerized anti-PACAP antibodies or antibody fragments thereof that maycomprise one or more of the following properties: (a) inhibit, block orprevent PACAP activation of at least one of PAC1 receptor (“PAC1-R”),vasoactive intestinal peptide receptor type 1 (“VPAC1-R”), and/orvasoactive intestinal peptide receptor type 2 (“VPAC2-R”); (b) inhibit,block or prevent PACAP activation of each of PAC1-R, VPAC1-R, andVPAC2-R; (c) inhibit, block or prevent PACAP activation of PAC1-R; (d)capable of inhibiting PACAP binding to at least one of PAC1-R, VPAC1-R,and/or VPAC2-R; (e) are capable of inhibiting PACAP binding to each ofPAC1-R, VPAC1-R, and/or VPAC2-R; (f) are capable of inhibiting PACAPbinding to PAC1-R-expressing cells; (g) are capable of inhibiting PACAPbinding to VPAC1-R-expressing cells; (h) may be capable of inhibitingPACAP binding to VPAC2-R-expressing cells; (i) inhibit PACAP binding tothe cell surface, e.g. via a glycosaminoglycan (“GAG”); (j) do notinhibit PACAP-mediated binding of such antibody to the cell surface,e.g., via a GAG; (k) inhibit PACAP-mediated binding of such antibody tothe cell surface, e.g., via a glycosaminoglycan (“GAG”); (1) inhibit,block or prevent PACAP-induced cAMP production; (m) when administered toa subject reduce PACAP-induced vasodilation; and/or (n) whenadministered to a subject reduces PACAP-induced photophobia, mast celldegranulation and/or neuronal activation.

The invention may also pertain to anti-PACAP antibodies and antigenbinding fragments thereof that may be preferably human, humanized orchimerized anti-PACAP antibodies or antibody fragments which may besubstantially non-immunogenic in human subjects. The invention may alsorelates to anti-PACAP antibodies and antigen binding fragments thereofthat may be preferably human, humanized or chimerized anti-PACAPantibodies or antibody fragments, which may be suitable for treating ahuman subject having an acute, episodic or chronic condition associatedwith increased vasodilation, photophobia, mast cell degranulation and/orneuronal activation.

Another embodiment of the invention relates to anti-PACAP antibodies andantigen binding fragments thereof that may be preferably human,humanized or chimerized anti-PACAP antibodies or antibody fragments thatspecifically bind to the same or overlapping linear or conformationalepitope(s) and/or may compete for binding to the same or overlappinglinear or conformational epitope(s) on human PACAP as an anti-PACAPantibody that may be selected from Ab10 or Ab20. Yet another embodimentof the invention relates to anti-PACAP antibodies and antigen bindingfragments thereof, preferably human, humanized or chimerized anti-PACAPantibodies or antibody fragments, that may specifically bind to the sameor overlapping linear or conformational epitope(s) on human PACAP as ananti-PACAP antibody that may be selected from Ab10 or Ab20. Said epitopeas described herein bound by said antibody or antibody fragment of theinvention may be identified by alanine scanning, e.g., as disclosed inExample 12 or another art-recognized method.

The invention may also embody anti-PACAP antibodies and antigen bindingfragments thereof that may be preferably human, humanized or chimerizedanti-PACAP antibodies or antibody fragments that may comprise at least 2complementarity determining regions (“CDRs”) of an anti-PACAP antibodythat may be selected from Ab10 or Ab20, preferably including the V_(H)CDR3 and/or the V_(L) CDR3. Another additional embodiment of theinvention may include anti-PACAP antibodies and antigen bindingfragments thereof that may be preferably human, humanized or chimerizedanti-PACAP antibodies or antibody fragments that may comprise at least3, at least 4, at least 5, or all 6 CDRS of an anti-PACAP antibody thatmay be selected from Ab10 or Ab20. In instances where all 6 CDRs may notbe present, preferably at least the V_(H) CDR3 and the V_(L) CDR3 may bepresent.

In another embodiment of the invention, anti-PACAP antibodies andantigen binding fragments thereof, preferably human, humanized orchimerized anti-PACAP antibodies or antibody fragments thereof, maycomprise a sequence variant of any of the antibodies or antibodyfragments of the invention that may contain one or more modificationsthat may putatively alter binding affinity or immunogenicity.

In a specific embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof according to the invention, are human, humanized, orchimerized anti-PACAP antibodies or antigen binding fragments thereof,and comprise (a) a variable heavy chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 404; a CDR2 sequence consisting of SEQ ID NO:406; and a CDR3 sequence consisting of SEQ ID NO: 408; and/or (b) avariable light chain comprising a CDR1 sequence consisting of SEQ ID NO:424; a CDR2 sequence consisting of SEQ ID NO: 426; and a CDR3 sequenceconsisting of SEQ ID NO: 428. Alternatively, the anti-PACAP antibodiesand antigen binding fragments thereof can comprise (a) a variable heavychain comprising an amino acid sequence with at least 80, 85, 90, 95,96, 97, 98, or 99% sequence identity to SEQ ID NO: 402, and/or (b) avariable light chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 422. Inanother embodiment, the anti-PACAP antibodies and antigen bindingfragments thereof comprise (a) a variable heavy chain having the aminoacid sequence of SEQ ID NO: 402, and/or (b) a variable light chainhaving the amino acid sequence of SEQ ID NO: 422. More specifically, theanti-PACAP antibodies and antigen binding fragments thereof can comprise(a) a heavy chain having the amino acid sequence of SEQ ID NO: 401,and/or (b) a light chain having the amino acid sequence of SEQ ID NO:421.

In another specific embodiment, the anti-PACAP antibodies and antigenbinding fragments thereof according to the invention, are human,humanized, or chimerized anti-PACAP antibodies or antigen bindingfragments thereof, and comprise (a) a variable heavy chain comprising aCDR1 sequence consisting of SEQ ID NO: 444; a CDR2 sequence consistingof SEQ ID NO: 446; and a CDR3 sequence consisting of SEQ ID NO: 448;and/or (b) a variable light chain comprising a CDR1 sequence consistingof SEQ ID NO: 464; a CDR2 sequence consisting of SEQ ID NO: 466; and aCDR3 sequence consisting of SEQ ID NO: 468. Alternatively, theanti-PACAP antibodies and antigen binding fragments thereof can comprise(a) a variable heavy chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:442, and/or (b) a variable light chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO: 462. In another embodiment, the anti-PACAP antibodies andantigen binding fragments thereof and comprise (a) a variable heavychain having the amino acid sequence of SEQ ID NO: 442, and/or (b) avariable light chain having the amino acid sequence of SEQ ID NO: 462.More specifically, the anti-PACAP antibodies and antigen bindingfragments thereof can comprise (a) a heavy chain having the amino acidsequence of SEQ ID NO: 441, and/or (b) a light chain having the aminoacid sequence of SEQ ID NO: 461.

Additionally, the anti-PACAP antibodies and antigen binding fragments ofthe invention may include human, humanized or chimerized anti-PACAPantibodies or antibody fragments wherein the antibodies or antibodyfragments may be selected from the group consisting of scFvs,camelbodies, nanobodies, Immunoglobulin New Antigen Receptor (“IgNAR”),fragment antigen binding (“Fab”) fragments, Fab′ fragments, MetMab likeantibodies, monovalent antibody fragments, and F(ab′)₂ fragments. Inanother embodiment, the anti-PACAP antibodies and antigen bindingfragments of the invention, preferably human, humanized or chimerizedanti-PACAP antibodies or antibody fragments, may substantially orentirely lack N-glycosylation and/or O-glycosylation. Also, theinvention embraces an embodiment of the invention wherein the anti-PACAPantibodies and antigen binding fragments of the invention, preferablyhuman, humanized or chimerized anti-PACAP antibodies or antibodyfragments, may comprise a human constant domain, e.g. that of an IgG1,IgG2, IgG3, or IgG4 antibody or fragment thereof.

An additional embodiment of the invention relates to anti-PACAPantibodies and antigen binding fragments, preferably human, humanized orchimerized anti-PACAP antibodies or antibody fragments, wherein saidantibodies or antibody fragments may comprise an Fc region that may havebeen modified to alter at least one of effector function, half-life,proteolysis, or glycosylation, e.g., wherein the Fc region may containone or more mutations that may alter or eliminate N- and/orO-glycosylation.

In yet another embodiment of the invention, anti-PACAP antibodies andantigen binding fragments, preferably human, humanized or chimerizedanti-PACAP antibodies or antibody fragments, may bind to PACAP with abinding affinity (K_(D)) of less than or equal to 5×10⁻⁵ M, 10⁻⁵ M,5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M,5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M,or 10⁻¹³ M, e.g., as may be determined by ELISA, bio-layerinterferometry (“BLI”), KINEXA or surface plasmon resonance at 25° or37° C. Also, another embodiment of the invention pertains to anti-PACAPantibodies and antigen binding fragments, preferably human, humanized orchimerized anti-PACAP antibodies or antibody fragments, wherein saidantibodies or antibody fragments may bind to PACAP with a bindingaffinity (K_(D)) of less than or equal to 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M,10⁻¹¹ M, 5×10⁻¹² M, or 10⁻¹² M. Additionally, the anti-PACAP antibodiesand antigen binding fragments, preferably human, humanized or chimerizedanti-PACAP antibodies or antibody fragments, of the invention mayinclude anti-PACAP antibodies or antibody fragments which may bind toPACAP with an off-rate (k_(off)) of less than or equal to 5×10⁻⁴ s⁻¹,10⁻⁴ s⁻¹, 5×10⁵ s⁻¹, or 10⁻⁵ s⁻¹.

Another embodiment of the invention relates to anti-PACAP antibodies andantigen binding fragments, preferably human, humanized or chimerizedanti-PACAP antibodies or antibody fragments, wherein said antibodies andantibody fragments may be directly or indirectly attached to adetectable label or therapeutic agent. An additional embodiment of theinvention pertains to anti-PACAP antibodies and antigen bindingfragments, preferably human, humanized or chimerized anti-PACAPantibodies or antibody fragments that when administered to a subject mayinhibit or neutralize at least one biological effect elicited by PACAP.The anti-PACAP antibodies and antigen binding fragments, preferablyhuman, humanized or chimerized anti-PACAP antibodies or antibodyfragments, of the invention may bind to PACAP27 and/or PACAP38 and mayblock PACAP27 and/or PACAP38 binding to PAC1-R, VPAC1-R, and/or VPAC2-R;may bind to PACAP27 and/or PACAP38 and may block PACAP27 and/or PACAP38binding to each of PAC1-R, VPAC1-R, and VPAC2-R; and/or may bind toPACAP27 and/or PACAP38 and may block PACAP27 and/or PACAP38 binding toPAC1-R-expressing cells, VPAC1-R-expressing cells, and/orVPAC2-R-expressing cells.

In another embodiment of the invention, the anti-PACAP antibodies andantigen binding fragments, preferably human, humanized or chimerizedanti-PACAP antibodies or antibody fragments, of the invention mayneutralize or inhibit PACAP activation of at least one of PAC1-R,VPAC1-R, or VPAC2-R; may neutralize or inhibit PACAP activation of eachof PAC1-R, VPAC1-R, and VPAC2-R; may neutralize or inhibit PACAPactivation of PAC1-R; may be capable of inhibiting or preventing PACAPbinding to at least one of PAC1-R, VPAC1-R, or VPAC2-R; may be capableof inhibiting or preventing PACAP binding to each of PAC1-R, VPAC1-R,and VPAC2-R; may be capable of inhibiting or preventing PACAP binding toPAC1-R-expressing cells, VPAC1-R-expressing cells, and/orVPAC2-R-expressing cells; may inhibit or block PACAP-induced cAMPproduction; may, when administered to a subject, reduce PACAP-inducedvasodilation, photophobia, mast cell degranulation and/or neuronalactivation.

In yet another embodiment of the invention, anti-PACAP antibodies andantigen binding fragments, preferably human, humanized or chimerizedanti-PACAP antibodies or antibody fragments, may bind to PACAP with aK_(D) that may be less than about 100 nM; with a K_(D) that may be lessthan about 40 nM; with a K_(D) that may be less than about 100 pM; witha K_(D) that may be less than about 50 pM; with a K_(D) that may be lessthan about 25 pM; or with a K_(D) that may be between about 10 pM andabout 100 pM. The invention also embraces anti-PACAP antibodies andantigen binding fragments, preferably human, humanized or chimerizedanti-PACAP antibodies or antibody fragments, that may have strongerbinding affinity for PACAP as compared to VIP and/or that may not bindto VIP, e.g., wherein said antibodies or antibody fragments thereof mayhave an affinity to PACAP that may be at least 10-fold, 30-fold,100-fold, 300-fold, 1000-fold, 3000-fold, 10000-fold, 30000-fold,100000-fold, 300000-fold, 1000000-fold, 3000000-fold, 10000000-fold,30000000-fold or more stronger than the affinity of said antibody orantibody fragment to VIP.

In another embodiment, the invention pertains to anti-PACAP antibodiesand antigen binding fragments, preferably human, humanized or chimerizedanti-PACAP antibodies or antibody fragments, that may be attached to atleast one effector moiety, e.g., wherein said effector moiety maycomprise a chemical linker. In another embodiment, the inventionpertains to anti-PACAP antibodies and antigen binding fragments,preferably human, humanized or chimerized anti-PACAP antibodies orantibody fragments that may be attached to one or more detectablemoieties, e.g., wherein said detectable moieties may comprise afluorescent dye, enzyme, substrate, bioluminescent material, radioactivematerial, chemiluminescent moiety, and/or mixtures thereof. Also, theanti-PACAP antibodies and antigen binding fragments of the invention,preferably human, humanized or chimerized anti-PACAP antibodies orantibody fragments, may be attached to one or more functional moieties.

Another embodiment of the invention relates to anti-idiotypic antibodiesthat may be produced against anti-PACAP antibodies or antibodyfragments, wherein said anti-idiotypic antibodies optionally mayneutralize one or more biological effects of the anti-PACAP antibody towhich it may bind. Said embodiment of the invention may also be relatedto a method of using said anti-idiotypic antibody to monitor the in vivolevels of said anti-PACAP antibody or antibody fragment in a subject orto neutralize the in vivo effects of said anti-PACAP antibody in asubject.

In yet another embodiment, the invention pertains to a composition thatmay be suitable for therapeutic, prophylactic, or a diagnostic use,whereby the composition may comprise a therapeutically, prophylacticallyor diagnostically effective amount of at least one anti-PACAP antibodyor antibody fragment or anti-idiotypic antibody, e.g., wherein thecomposition may be suitable for administration via injection, topical,oral, inhalation or transdermal; may be suitable for subcutaneous,intravenous, intramuscular, topical, oral, inhalatory, intranasal,intrabuccal, vaginal, anal, transdermal, intraperitoneal, or intrathecaladministration; and/or wherein the composition may be suitable forsubcutaneous intravenous or intramuscular administration. The inventionalso embraces an embodiment of the invention wherein said composition ofat least one anti-PACAP antibody or antibody fragment or anti-idiotypicantibody may be lyophilized; and/or wherein said composition maycomprise a pharmaceutically acceptable diluent, carrier, solubilizer,emulsifier, preservative, or mixture thereof. Said composition of theinvention may further comprise at least one other active agent, e.g.,wherein the other active agent may be selected from the group consistingof a chemotherapeutic, an analgesic, an anti-inflammatory, animmunosuppressant, a cytokine, an antiproliferative, an antiemeticand/or a cytotoxin. Said composition may also be lyophilized,stabilized, and/or formulated for administration by injection.

A further embodiment of the invention embraces an isolated nucleic acidsequence or nucleic acid sequences that may encode an anti-PACAPantibody or antibody fragment or anti-idiotypic antibody, and whereinsaid isolated nucleic acid sequence or nucleic acid sequences may becontained within a vector or vectors. Additionally, in an embodiment ofthe invention, a host cell may comprise said isolated nucleic acidsequence or sequences, wherein said host cell may be a mammalian,bacterial, fungal, yeast, avian, amphibian, plant or insect cell; and/orsaid host cell may be a filamentous fungus or a yeast. Wherein said hostcell may be a yeast cell, the yeast may be selected from the followinggenera: Arxiozyma; Ascobotryozyma; Citeromyces; Debaryomyces; Dekkera;Eremothecium; Issatchenkia; Kazachstania; Kluyveromyces; Kodamaea;Lodderomyces; Pachysolen; Pichia; Saccharomyces; Saturnispora;Tetrapisispora; Torulaspora; Williopsis; and Zygosaccharomyces; or theyeast may be genus Pichia. In an embodiment of the invention, the yeasthost cell may be selected from Pichia pastoris, Pichia methanolica andHansenula polymorpha (Pichia angusta). Additionally, said host cell maybe a filamentous fungus or a yeast or may be a CHO cell.

The invention also relates to a method of expressing an anti-PACAPantibody or antibody fragment that may comprise culturing any of but notlimited to the host cells disclosed herein under conditions that mayprovide for expression of said antibody or antibody fragment.Additionally, the invention pertains to said method wherein the hostcell may be a yeast cell or CHO cell that may stably express and secretesaid antibody or antibody fragment. For example, said yeast cell may bea polyploid yeast that may be made by a method that may comprise: (i)introducing at least one expression vector containing one or moreheterologous polynucleotides encoding said antibody operably linked to apromoter and a signal sequence into a haploid yeast cell; (ii) producingby mating or spheroplast fusion a polyploid yeast from said first and/orsecond haploid yeast cell; (iii) selecting polyploid yeast cells thatstably express said antibody; and (iv) producing stable polyploid yeastcultures from said polyploid yeast cells that stably express saidantibody into the culture medium; and said polyploid yeast may be of thegenus Pichia. Additionally in another embodiment, the invention embracesa method of expressing an anti-PACAP antibody or antibody fragment thatmay comprise culturing a host cell wherein said host cell may be amammalian cell, e.g., a CHO cell.

Additionally, the invention pertains to a method that may block,inhibit, block or neutralize one or more biological effects associatedwith PACAP in a subject that may comprise administering to said subjecta therapeutically or prophylactically effective amount of a human,humanized or chimerized anti-PACAP antibody or antibody fragment thatmay antagonize, inhibit, neutralize or block at least one biologicaleffect associated with human PACAP. Another aspect of the inventionrelates to a method that may block, inhibit, or neutralize one or morebiological effects associated with PACAP in a subject that may compriseadministering to said subject a therapeutically or prophylacticallyeffective amount of a human, humanized or chimerized anti-PACAP antibodyor antibody fragment that may antagonize, inhibit, neutralize or blockat least one biological effect associated with human PACAP and that maynot substantially interact with (bind) VIP.

Another aspect of the invention generally relates to a method that mayblock, inhibit, or neutralize one or more biological effects, e.g.,vasomotor effects, associated with PACAP in a subject that may compriseadministering to a subject a therapeutically or prophylacticallyeffective amount of a human, humanized or chimerized anti-PACAP antibodyor antibody fragment that may comprise one or more of the following:inhibits, blocks or neutralizes at least one biological effect elicitedby PACAP; neutralizes or inhibits PACAP activation of at least one ofPAC1-R, VPAC1-R, and/or VPAC2-R; inhibits, blocks or neutralizes PACAPactivation of each of PAC1-R, VPAC1-R, and VPAC2-R; neutralizes orinhibits PACAP activation of PAC1-R; inhibits PACAP binding to at leastone of PAC1-R, VPAC1-R, and/or VPAC2-R; inhibits PACAP binding to eachof PAC1-R, VPAC1-R, and/or VPAC2-R; inhibits PACAP binding toPAC1-R-expressing cells; inhibits PACAP binding to VPAC1-R and/orVPAC2-R-expressing cells; does not inhibit PACAP-mediated binding ofsuch antibody to the cell surface, e.g., via a glycosaminoglycan(“GAG”); inhibits PACAP binding to the cell surface, e.g. via aglycosaminoglycan (“GAG”); inhibits PACAP-induced cAMP production;and/or, when administered to a subject, may reduce PACAP-inducedvasodilation, photophobia, mast cell degranulation and/or neuronalactivation.

Another embodiment of the invention relates to method that may block,inhibit, or neutralize vasodilation, e.g., vasodilation of the duralarteries, which may be associated with or may be elicited by PACAP in asubject that may comprise administering to a subject a therapeuticallyor prophylactically effective amount of a human, humanized or chimerizedanti-PACAP antibody or antibody fragment that may block, inhibit, orneutralize vasodilation associated with, or elicited by PACAP.

Yet another embodiment of the invention pertains to a method that maytreat or prevent the onset, frequency, severity or duration of headacheor migraine, e.g., wherein said headache or migraine may be selectedfrom migraine with aura, migraine without aura, hemiplegic migraine,cluster headache, migrainous neuralgia, chronic headache, chronicmigraine, medication overuse headache, and tension headache, in asubject that may comprise administering to a subject in need thereof aneffective amount of a human, humanized or chimerized anti-human PACAPantibody or antibody fragment that may elicit one or more of thefollowing effects: inhibits or neutralizes at least one biologicaleffect elicited by PACAP; neutralizes or inhibits PACAP activation of atleast one of PAC1-R, VPAC1-R, and/or VPAC2-R; neutralizes or inhibitsPACAP activation of each of PAC1-R, VPAC1-R, and VPAC2-R; neutralizes orinhibits PACAP activation of PAC1-R; inhibits PACAP binding to at leastone of PAC1-R, VPAC1-R, and/or VPAC2-R; inhibits PACAP binding to eachof PAC1-R, VPAC1-R, and/or VPAC2-R; inhibits PACAP binding toPAC1-R-expressing cells; inhibits PACAP binding to VPAC1-R and/orVPAC2-R-expressing cells; inhibits PACAP-mediated binding of suchantibody to the cell surface, e.g., via a GAG; inhibits PACAP binding tothe cell surface, e.g. via a glycosaminoglycan (“GAG”); inhibitsPACAP-induced cyclic adenosine monophosphate (“cAMP”) production;and/or, when administered to a subject, reduces PACAP-inducedvasodilation, photophobia, mast cell degranulation and/or neuronalactivation.

Another embodiment of the invention relates to a method of treating ahuman subject that may have an acute, episodic or chronic condition thatmay be associated with at least one of increased vasodilation,photophobia, mast cell degranulation and neuronal activation or acombination of said conditions that may comprise administering to asubject in need thereof an effective amount of an antagonistic human,humanized or chimerized anti-human PACAP antibody or antibody fragment.

In yet another embodiment, the invention relates to a method that mayblock, inhibit, or neutralize one or more biological effects associatedwith PACAP in a subject that may comprise administering to a subject inneed thereof an effective amount of a human, humanized or chimerizedanti-PACAP antibody or antigen binding fragment that may antagonize,inhibit, neutralize, or block at least one biological effect associatedwith human PACAP. Also, an embodiment of the invention encompasses amethod that may block, inhibit, or neutralize one or more biologicaleffects associated with PACAP in a subject that may compriseadministering to a subject in need thereof an effective amount of ahuman, humanized, or chimerized anti-PACAP antibody or antigen bindingfragment that may antagonize, inhibit, neutralize, or block at least onebiological effect associated with human PACAP and that may notsubstantially interact with (bind) VIP.

In another embodiment, the invention relates to a method that may block,inhibit, or neutralize one or more biological effects associated withPACAP in a subject that may comprise administering to a subject in needthereof an effective amount of a human, humanized, or chimerizedanti-PACAP antibody or antigen binding fragment that (a) inhibits orneutralizes at least one biological effect elicited by PACAP; (b)neutralizes or inhibits PACAP activation of at least one of PAC1receptor (“PAC1-R”), vasoactive intestinal peptide receptor type 1(“VPAC1-R”), and/or vasoactive intestinal peptide receptor type 2(“VPAC2-R”); (c) neutralizes or inhibits PACAP activation of each ofPAC1-R, VPAC1-R, and VPAC2-R; (d) neutralizes or inhibits PACAPactivation of PAC1-R; (e) inhibits PACAP binding to at least one ofPAC1-R, VPAC1-R, and/or VPAC2-R; (f) inhibits PACAP binding to each ofPAC1-R, VPAC1-R, and/or VPAC2-R; (g) may be capable of inhibiting PACAPbinding to PAC1-R-expressing cells; (h) inhibitis PACAP binding to thecell surface, e.g., via a glycosaminoglycan (“GAG”) (i) inhibitsPACAP-mediated binding of such antibody to the cell surface, e.g, via aGAG; (j) inhibits PACAP-induced cyclic adenosine monophosphate (“cAMP”)production; and/or (k) when administered to the subject reducesPACAP-induced vasodilation, photophobia, mast cell degranulation and/orneuronal activation.

Moreover, an embodiment of the invention pertains to a method oftreating or preventing the onset, frequency, severity or duration ofheadache or migraine, e.g., wherein the headache or migraine may beselected from migraine with aura, migraine without aura, hemiplegicmigraine, cluster headache, migrainous neuralgia, chronic headache,chronic migraine, medication overuse headache, and tension headache, ina subject that may comprise administering to a subject in need thereofan effective amount of a human, humanized, or chimerized anti-humanPACAP antibody or antigen binding fragment that (a) inhibits orneutralize at least one biological effect elicited by PACAP; (b)neutralizes or inhibits PACAP activation of at least one of PAC1receptor (“PAC1-R”), vasoactive intestinal peptide receptor type 1(“VPAC1-R”), and/or vasoactive intestinal peptide receptor type 2(“VPAC2-R”); (c) neutralizes or inhibits PACAP activation of each ofPAC1-R, VPAC1-R, and VPAC2-R; (d) neutralizes or inhibits PACAPactivation of PAC1-R; (e) may be capable of inhibiting PACAP binding toat least one of PAC1-R, VPAC1-R, and/or VPAC2-R; (f) inhibits PACAPbinding to each of PAC1-R, VPAC1-R, and/or VPAC2-R; (g) inhibits PACAPbinding to PAC1-R-expressing cells; (h) inhibits PACAP binding to thecell surface, e.g., via a glycosaminoglycan (“GAG”); (i) inhibitsPACAP-mediated binding of such antibody to the cell surface, e.g, via aGAG; (j) inhibits PACAP-induced cyclic adenosine monophosphate (“cAMP”)production; and/or (k) when administered to the subject reducesPACAP-induced vasodilation, photophobia, mast cell degranulation and/orneuronal activation.

A further embodiment of the invention encompasses a method of treating ahuman subject that may have an acute, episodic or chronic conditionassociated with at least one of increased vasodilation, photophobia,mast cell degranulation and neuronal activation or a combination of anyof above that may comprise administering to a subject in need thereof aneffective amount of an antagonistic human, humanized or chimerizedanti-human PACAP antibody or antigen binding fragment.

The invention also pertains to any of the methods disclosed herein thatmay include the administration of anti-PACAP antibody or antigen bindingfragment of the invention, and wherein said anti-PACAP antibody may be ahuman antibody or antigen binding fragment; and/or wherein theanti-PACAP antibody may be a humanized antibody or antigen bindingfragment; and/or wherein the anti-PACAP antibody may be a chimericantibody or antigen binding fragment.

Another embodiment of the invention also relates to a method wherein ananti-PACAP antibody or antibody fragment of the invention may bind toPACAP27 and/or PACAP38 and may block PACAP27 and/or PACAP38 binding toPAC1-R, VPAC1-R, and/or VPAC2-R. Another embodiment of the inventionpertains to a method wherein said anti-PACAP antibody or antibodyfragment may bind to PACAP27 and/or PACAP38 and may block PACAP27 and/orPACAP38 binding to each of PAC1-R, VPAC1-R, and VPAC2-R. Yet anotherembodiment of the invention relates to a method wherein said anti-PACAPantibody or antibody fragment may bind to PACAP27 and/or PACAP38 and mayblock PACAP27 and/or PACAP38 binding to PAC1-R-expressing cells.Additionally, said anti-PACAP antibody or antibody fragment of theinvention may have an affinity to PACAP that may be at least 10-fold,30-fold, 100-fold, 300-fold, 1000-fold, 3000-fold, 10000-fold,30000-fold, 100000-fold, 300000-fold, 1000000-fold, 3000000-fold,10000000-fold, 30000000-fold or more stronger than the affinity of saidantibody or antibody fragment to VIP.

The invention embraces a method that may block, inhibit, block orneutralize one or more biological effects associated with PACAP in asubject that may comprise administering to said subject atherapeutically or prophylactically effective amount of a human,humanized or chimerized anti-PACAP antibody or antibody fragment thatmay antagonize, inhibit, neutralize or blocks at least one biologicaleffect associated with human PACAP, and wherein said subject may have acondition selected from the group consisting of migraine with aura,migraine without aura, hemiplegic migraines, cluster headaches,migrainous neuralgia, chronic headaches, tension headaches, generalheadaches, hot flush, photophobia, chronic paroxysmal hemicrania,secondary headaches due to an underlying structural problem in the head,secondary headaches due to an underlying structural problem in the neck,cranial neuralgia, sinus headaches, headache associated with sinusitis,allergy-induced headaches, allergy-induced migraines, trigeminalneuralgia, post-herpetic neuralgia, phantom limb pain, fibromyalgia,reflex sympathetic dystrophy, pain, chronic pain, inflammatory pain,post-operative incision pain, post-surgical pain, trauma-related pain,lower back pain, eye pain, tooth pain, complex regional pain syndrome,cancer pain, primary or metastatic bone cancer pain, fracture pain,osteoporotic fracture pain, pain resulting from burn, gout joint pain,pain associated with sickle cell crises, pain associated withtemporomandibular disorders, cirrhosis, hepatitis, neurogenic pain,neuropathic pain, nociceptic pain, visceral pain, menstrual pain,ovarialgia, osteoarthritis pain, rheumatoid arthritis pain, diabeticneuropathy, sciatica, dyspepsia, irritable bowel syndrome, inflammatorybowel disease, Crohn's disease, ileitis, ulcerative colitis, renalcolic, dysmenorrhea, cystitis, interstitial cystitis, menstrual period,labor, menopause, pancreatitis, schizophrenia, depression,post-traumatic stress disorder (“PTSD”), anxiety disorders, autoimmunediabetes, Sjögren's syndrome, multiple sclerosis, overactive bladder,bronchial hyperreactivity, asthma, stroke, bronchitis, bronchodilation,emphysema, chronic obstructive pulmonary disease (“COPD”), inflammatorydermatitis, adenocarcinoma in glandular tissue, blastoma in embryonictissue of organs, carcinoma in epithelial tissue, leukemia in tissuesthat form blood cells, lymphoma in lymphatic tissue, myeloma in bonemarrow, sarcoma in connective or supportive tissue, adrenal cancer,AIDS-related lymphoma, anemia, bladder cancer, bone cancer, braincancer, breast cancer, carcinoid tumors, cervical cancer, chemotherapy,colon cancer, cytopenia, endometrial cancer, esophageal cancer, gastriccancer, head cancer, neck cancer, hepatobiliary cancer, kidney cancer,leukemia, liver cancer, lung cancer, lymphoma, Hodgkin's disease,non-Hodgkin's, nervous system tumors, oral cancer, ovarian cancer,pancreatic cancer, prostate cancer, rectal cancer, skin cancer, stomachcancer, testicular cancer, thyroid cancer, urethral cancer, cancer ofbone marrow, multiple myeloma, tumors that metastasize to the bone,tumors infiltrating the nerve and hollow viscus, tumors near neuralstructures, acne vulgaris, atopic dermatitis, urticaria, keloids,hypertrophic scars and rosacea, endothelial dysfunction, Raynaud'ssyndrome, coronary heart disease (“CHD”), coronary artery disease(“CAD”), heart failure, peripheral arterial disease (“PAD”), diabetes,pulmonary hypertension (“PH”), connective tissue disorder, allergicdermatitis, psoriasis, pruritus, neurogenic cutaneous redness, erythema,sarcoidosis, shock, sepsis, opiate withdrawal syndrome, morphinetolerance, and epilepsy. Additionally, said subject may have a conditionselected from the group consisting of migraine, headache and a painassociated disease or condition, wherein said headache or migraine mayselected from the group consisting of migraine with aura, migrainewithout aura, hemiplegic migraine, cluster headache, migrainousneuralgia, chronic headache, chronic migraine, medication overuseheadache, and tension headache. Also, said subject may have a oculardisorder associated with photophobia selected from the group consistingof achromatopsia, aniridia, photophobia caused by an anticholinergicdrug, aphakia, buphthalmos, cataracts, cone dystrophy, congenitalabnormalities of the eye, viral conjunctivitis, corneal abrasion,corneal dystrophy, corneal ulcer, disruption of the corneal epithelium,ectopia lentis, endophthalmitis, eye trauma caused by disease, eyetrauma caused by injury, eye trauma caused by infection, chalazion,episcleritis, glaucoma, keratoconus, optic nerve hypoplasia,hydrophthalmos, congenital glaucoma iritis, optic neuritis, pigmentdispersion syndrome, pupillary dilation, retinal detachment, scarring ofthe cornea, sclera and uveitis. Further, said subject may have a nervoussystem-related or neurological condition associated with photophobiaselected from the group consisting of autism spectrum disorders, Chiarimalformation, dyslexia, encephalitis, meningitis, subarachnoidhemorrhage, tumor of the posterior cranial fossa, ankylosingspondylitis, albinism, ariboflavinosis, benzodiazepines, chemotherapy,chikungunya, cystinosis, Ehlers-Danlos syndrome, hangover, influenza,infectious mononucleosis, magnesium deficiency, mercury poisoning,migraine, rabies, and tyrosinemia type II. Additionally, said subjectmay have a photophobia associated disorder selected from the groupconsisting of migraine with aura, migraine without aura, iritis,uveitis, meningitis, depression, bipolar disorder, cluster headache oranther trigeminal autonomic cephalalgia (“TAC”) or blepharospasm,depression, agoraphobia and bipolar disorder.

The invention additionally embraces any of the methods disclosed hereinwherein an antibody of any of the methods disclosed herein may be ahuman, humanized, or chimerized anti-PACAP antibody or antigen bindingfragment, and/or wherein the epitope bound by said antibody of any ofthe methods disclosed herein may be identified by alanine scanning.

The invention also embraces any of the methods disclosed herein whereinthe method relates to an antibody or antibody fragment that may be ahuman, humanized, or chimerized anti-PACAP antibody or antibodyfragment; and/or wherein the antibody or antibody fragment may be ahuman, humanized, or chimerized anti-PACAP antibody or antibodyfragment; and/or wherein the antibody or antibody fragment may be ananti-PACAP antibody or antibody fragment that may comprise the same CDRsas an anti-PACAP antibody that may be selected from Ab10 or Ab20.

The invention additionally embraces any of the methods disclosed hereinwherein the anti-PACAP antibody or antigen binding fragment may compriseat least 3 CDRs; at least 4 CDRs; at least 5 CDRs; or all 6 CDRs of ananti-PACAP antibody that may be selected from Ab10 or Ab20.

The invention also embraces any of the methods disclosed herein whereinthe anti-PACAP antibodies and antigen binding fragments thereofaccording to the invention, are human, humanized, or chimerizedanti-PACAP antibodies or antigen binding fragments thereof, and comprise(a) a variable heavy chain comprising a CDR1 sequence consisting of SEQID NO: 404; a CDR2 sequence consisting of SEQ ID NO: 406; and a CDR3sequence consisting of SEQ ID NO: 408; and/or (b) a variable light chaincomprising a CDR1 sequence consisting of SEQ ID NO: 424; a CDR2 sequenceconsisting of SEQ ID NO: 426; and a CDR3 sequence consisting of SEQ IDNO: 428. Alternatively, the anti-PACAP antibodies and antigen bindingfragments thereof can comprise (a) a variable heavy chain comprising anamino acid sequence with at least 80, 85, 90, 95, 96, 97, 98, or 99%sequence identity to SEQ ID NO: 402, and/or (b) a variable light chaincomprising an amino acid sequence with at least 80, 85, 90, 95, 96, 97,98, or 99% sequence identity to SEQ ID NO: 422. In another embodiment,the anti-PACAP antibodies and antigen binding fragments thereof comprise(a) a variable heavy chain having the amino acid sequence of SEQ ID NO:402, and/or (b) a variable light chain having the amino acid sequence ofSEQ ID NO: 422. More specifically, the anti-PACAP antibodies and antigenbinding fragments thereof can comprise (a) a heavy chain having theamino acid sequence of SEQ ID NO: 401, and/or (b) a light chain havingthe amino acid sequence of SEQ ID NO: 421.

The invention also embraces any of the methods disclosed herein whereinthe anti-PACAP antibodies and antigen binding fragments thereofaccording to the invention, are human, humanized, or chimerizedanti-PACAP antibodies or antigen binding fragments thereof, and comprise(a) a variable heavy chain comprising a CDR1 sequence consisting of SEQID NO: 444; a CDR2 sequence consisting of SEQ ID NO: 446; and a CDR3sequence consisting of SEQ ID NO: 448; and/or (b) a variable light chaincomprising a CDR1 sequence consisting of SEQ ID NO: 464; a CDR2 sequenceconsisting of SEQ ID NO: 466; and a CDR3 sequence consisting of SEQ IDNO: 468. Alternatively, the anti-PACAP antibodies and antigen bindingfragments thereof can comprise (a) a variable heavy chain comprising anamino acid sequence with at least 80, 85, 90, 95, 96, 97, 98, or 99%sequence identity to SEQ ID NO: 442, and/or (b) a variable light chaincomprising an amino acid sequence with at least 80, 85, 90, 95, 96, 97,98, or 99% sequence identity to SEQ ID NO: 462. In another embodiment,the anti-PACAP antibodies and antigen binding fragments thereof andcomprise (a) a variable heavy chain having the amino acid sequence ofSEQ ID NO: 442, and/or (b) a variable light chain having the amino acidsequence of SEQ ID NO: 462. More specifically, the anti-PACAP antibodiesand antigen binding fragments thereof can comprise (a) a heavy chainhaving the amino acid sequence of SEQ ID NO: 441, and/or (b) a lightchain having the amino acid sequence of SEQ ID NO: 461.

The invention also relates to any of the methods disclosed hereinwherein the anti-PACAP antibodies or antibody fragments may be selectedfrom the group consisting of scFvs, camelbodies, nanobodies,Immunoglobulin New Antigen Receptor (“IgNAR”), fragment antigen binding(“Fab”) fragments, Fab′ fragments, MetMab like antibodies, monovalentantibody fragments, and F(ab′)₂ fragments. Additionally, the inventionrelates to any of the methods disclosed herein wherein the anti-PACAPantibody or antibody fragment may substantially or entirely lackN-glycosylation and/or O-glycosylation. Also, the invention pertains toany of the methods disclosed herein wherein the anti-PACAP antibody orantibody fragment may comprise a human constant domain, e.g., that of anIgG1, IgG2, IgG3, or IgG4 antibody.

Another aspect of the invention pertains to any of the methods disclosedherein wherein the anti-PACAP antibody or antibody fragment may comprisean Fc region that may have been modified to alter at least one ofeffector function, half-life, proteolysis, or glycosylation, e.g., theFc region may contain one or more mutations that may alter or eliminateN- and/or O-glycosylation.

A further aspect of the invention relates to any of the methodsdisclosed herein wherein the anti-PACAP antibody or antibody fragmentmay bind to PACAP with a binding affinity (K_(D)) of less than or equalto 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸M, 10⁻⁸M, 5×10⁻⁹M, 10⁻⁹M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M,10⁻¹² M, 5×10⁻¹³ M, or 10⁻¹³ M. Also, said anti-PACAP antibody orantibody fragment of any of the methods disclosed herein may bind toPACAP with a binding affinity (K_(D)) of less than or equal to 5×10¹⁰ M,10¹⁰ M, 5×10¹¹ M, 10¹¹ M, 5×10¹² M, or 10¹² M. Another embodiment of theinvention pertains any of the methods disclosed herein wherein theanti-PACAP antibody or antibody fragment may bind to PACAP with anoff-rate (k_(off)) of less than or equal to 5×10⁻⁴ s⁻¹, 10⁻⁴ s⁻¹, 5×10⁻⁵s⁻¹, or 10⁻⁵ s⁻¹.

Moreover, the invention embraces any of the methods disclosed hereinwherein the anti-PACAP antibody or antibody fragment may be directly orindirectly attached to a detectable label or therapeutic agent. Also,the invention relates to any of the methods disclosed herein wherein theanti-PACAP antibody or antibody fragment may bind to PACAP with a K_(D)that may be less than about 100 nM, less than about 40 nM, less thanabout 1 nM, less than about 100 pM, less than about 50 pM, or less thanabout 25 pM. Also, the invention embraces any of the methods disclosedherein wherein the anti-PACAP antibody or antibody fragment may bind toPACAP with a K_(D) that may be between about 10 pM and about 100 pM. Theinvention further pertains to any of the methods disclosed hereinwherein the method may further comprise administering separately orco-administering another agent, e.g., wherein the other agent may beselected from a chemotherapeutic, an analgesic, an anti-inflammatory, animmunosuppressant, a cytokine, an antiproliferative, an antiemetic or acytotoxin. Also, the invention embraces any of the methods disclosedherein wherein the other therapeutic agent may be an analgesic, and saidanalgesic may be a non-steroidal anti-inflammatory drug (“NSAID”), anopioid analgesic, another antibody or a non-antibody biologic, andfurther wherein said other antibody may be an anti-NGF antibody orantibody fragment; and/or may be an anti-Calcitonin Gene-Related Peptide(“CGRP”) antibody or antibody fragment and/or an anti-CGRP receptorantibody or antibody fragment. The invention also pertains to any of themethods disclosed herein wherein said NSAID may be a cyclooxygenase 1and/or cyclooxygenase 2 inhibitor.

In another embodiment of the invention, an anti-PACAP antibody orantibody fragment or a method as disclosed herein may inhibit theeffects of PACAP on vasodilation; and/or may inhibit the effects ofPACAP on cAMP production; and/or may inhibit the effects of PACAP on PLCresulting in reduced Ca⁺⁺ and PLD levels; and/or may inhibit the effectsof PACAP on adenylate cyclase activity; and/or may inhibit the effectsof PACAP on its binding to any or all of PAC1-R, VPAC1-R or VPAC2-R;and/or may inhibit the effects of PACAP on neurodevelopment; may inhibitthe effects of PACAP on neuroprotection; and/or may inhibit the effectsof PACAP on neuromodulation; and/or may inhibit the effects of PACAP onneurogenic inflammation; and/or may inhibit the effects of PACAP onnociception; and/or may modulate the interaction of PACAP with bindingthe cell surface, e.g. via at least one GAG, e.g., wherein at least onesaid GAG may comprise one or more of heparin, chondroitin, keratin, andhyaluronic acid, and further wherein said antibody or antibody fragmentor method may block or inhibit receptor-independent cellular uptake ofPACAP38 and/or PACAP27 and/or may inhibit or may block GAG-dependentuptake of PACAP38 and/or PACAP27 by cells.

A further embodiment of the invention relates to method of therapy orprophylaxis that may comprise the administration of an anti-PACAPantibody or antibody fragment of the invention. Additionally, theinvention pertains to a composition that may be used in human therapythat may contain an anti-PACAP antibody or antibody fragment of theinvention. Said composition may contain another active agent, e.g.,wherein the other agent may be selected from a chemotherapeutic, ananalgesic, an anti-inflammatory, an immunosuppressant, a cytokine, anantiproliferative, an antiemetic or a cytotoxin. Wherein said otheragent may be an analgesic, said analgesic may be a NSAID, an opioidanalgesic, another antibody or a non-antibody biologic. When said otheragent may be an analgesic that may be another antibody, the otherantibody may be an anti-NGF antibody or antibody fragment, and/or theother antibody may be an anti-Calcitonin Gene-Related Peptide (“CGRP”)antibody or antibody fragment and/or an anti-CGRP receptor antibody orantibody fragment. Wherein said other agent may be a NSAID, said NSAIDmay be a cyclooxygenase 1 and/or cyclooxygenase 2 inhibitor; and/or saidNSAID may be selected from the group consisting of (1) propionic acidderivatives including ibuprofen, naproxen, naprosyn, diclofenac, andketoprofen; (2) acetic acid derivatives including tolmetin and sulindac;(3) fenamic acid derivatives including mefenamic acid and meclofenamicacid; (4) biphenylcarboxylic acid derivatives including diflunisal andflufenisal; and (5) oxicams including piroxim, sudoxicam, and isoxicam.Wherein said other agent may be an opioid analgesic, said opioidanalgesic may be selected from the group consisting of codeine,dihydrocodeine, diacetylmorphine, hydrocodone, hydromorphone,levorphanol, oxymorphone, alfentanil, buprenorphine, butorphanol,fentanyl, sufentanil, meperidine, methadone, nalbuphine, propoxyphene,pentazocine, and pharmaceutically acceptable salts thereof; and/or saidopioid analgesic may be morphine or a morphine derivative orpharmaceutically acceptable salt thereof; and/or said opioid analgesicand an anti-PACAP antibody or antigen binding fragment according to theinvention may increase the analgesic effect as compared to either theopioid analgesic or the anti-PACAP antibody or antigen binding fragmentadministered alone.

In another embodiment of the invention, an anti-PACAP antibody orfragment or composition according to the invention, wherein theanti-PACAP antibody or fragment and another active agent may be combinedtherewith or may be administered in combination, may elicit asynergistic or additive effect on the treatment or prevention of a PACAPassociated effect, e.g., migraine or on pain. The invention additionallyembraces the anti-PACAP antibody or fragment or composition accordingthe invention that may be used in therapy or diagnosis, e.g., migrainetreatment or prophylaxis. A further embodiment of the invention relatesto anti-PACAP antibody or fragment or composition according to theinvention that may be used for potentially treating one or more of hotflush, migraine with or without aura, hemiplegic migraine, clusterheadache, migrainous neuralgia, chronic headache, chronic migraine,medication overuse headache or tension headache.

Another embodiment of the invention pertains anti-PACAP antibody orfragment or composition according to the invention that may be used forameliorating, controlling, reducing incidence of, or delaying thedevelopment or progression of headache, e.g., migraine with or withoutaura, hemiplegic migraine, cluster headache, migrainous neuralgia,chronic headache, chronic migraine, medication overuse headache ortension headache. In an embodiment of the invention, the use may be fora subject who may have previously received or may be receiving ananti-CGRP antibody or antibody fragment and/or an anti-CGRP receptorantibody or antibody fragment, and further wherein said subject may be amigraineur who may not have adequately responded to anti-CGRP antibodyor antibody fragment and/or anti-CGRP receptor antibody or antibodyfragment treatment; and/or wherein said subject may have previouslyreceived at least one anti-CGRP antibody or antibody fragment and/oranti-CGRP receptor antibody or antibody fragment administration that mayhave elicited an immune response to the anti-CGRP antibody or antibodyfragment and/or anti-CGRP receptor antibody or antibody fragment.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

FIG. 1A-1B provide the polypeptide sequences of the variable heavy chainregion for antibodies Ab10, Ab20, and Ab21, Ab22, and Ab23 (SEQ ID NO:402; 442; 842; 882; and 922, respectively) aligned by their FRs andCDRs.

FIG. 2A-2B provide the polypeptide sequences of the variable light chainregion for antibodies Ab10, Ab20, Ab21, Ab22, and Ab23 (SEQ ID NO: 422;462; 862; 902; and 942, respectively) aligned by their FRs and CDRs.

FIG. 3A-3F provide the polynucleotide sequences encoding the variableheavy chain region for antibodies Ab10, Ab20, Ab21, Ab22, and Ab23 (SEQID NO: 412; 452; 852; 892; and 932, respectively) aligned by their FRsand CDRs.

FIG. 4A-4E provide the polynucleotide sequences encoding the variablelight chain region for antibodies Ab10, Ab20, Ab21, Ab22, and Ab23 (SEQID NO: 432; 472; 872; 912; and 952, respectively) aligned by their FRsand CDRs.

FIG. 5 provides the polypeptide sequence coordinates for certainantibody heavy chain protein sequence features including the variableregion and CDRs of the heavy chain for antibodies Ab10, Ab20, Ab21,Ab22, and Ab23.

FIG. 6 provides the polypeptide sequence coordinates for certainantibody heavy chain protein sequence features including the constantregion and framework regions FRs of the heavy chain for antibodies Ab10,Ab20, Ab21, Ab22, and Ab23.

FIG. 7 provides the polypeptide sequence coordinates for certainantibody light chain protein sequence features including the variableregion and CDRs of the light chain for antibodies Ab10, Ab20, Ab21,Ab22, and Ab23.

FIG. 8 provides the polypeptide sequence coordinates for certainantibody light chain protein sequence features including the constantregion and framework regions FRs of the light chain for antibodies Ab10,Ab20, Ab21, Ab22, and Ab23.

FIG. 9 provides the polynucleotide sequence coordinates for certainantibody heavy chain DNA sequence features including the variable regionand CDRs of the heavy chain for antibodies Ab10, Ab20, Ab21, Ab22, andAb23.

FIG. 10 provides the polynucleotide sequence coordinates for certainantibody heavy chain DNA sequence features including the constant regionand FRs of the heavy chain for antibodies Ab10, Ab20, Ab21, Ab22, andAb23.

FIG. 11 provides the polynucleotide sequence coordinates for certainantibody light chain DNA sequence features including the variable regionand CDRs of the light chain for antibodies Ab10, Ab20, Ab21, Ab22, andAb23.

FIG. 12 provides the polynucleotide sequence coordinates for certainantibody light chain DNA sequence features including the constant regionand FRs of the light chain for antibodies Ab10, Ab20, Ab21, Ab22, andAb23.

FIG. 13A-F provides representative competitive binding data for Ab10(FIG. 13A), Ab20 (FIG. 13B), Ab21 (FIG. 13C), Ab1.H (FIG. 13D), Ab22(FIG. 13E), and Ab23 (FIG. 13F) obtained following the protocol inExample 1 infra.

FIG. 14A-H provides representative data showing Ab10-mediated (FIG.14A), Ab20-mediated (FIG. 14B), Ab21-mediated (FIG. 14C), Ab1.H-mediated(FIG. 14D), Ab10.H-mediated (FIG. 14E), Ab21.H-mediated (FIG. 14F),Ab22-mediated (FIG. 14G), and Ab23-mediated (FIG. 14H) inhibition ofPACAP38 binding to PAC1-R-expressing PC-12 cells obtained following theprotocol in Example 5 infra.

FIG. 15A-H provides representative data showing Ab10 (FIG. 15A), Ab20(FIG. 15B), Ab21 (FIG. 15C), Ab1.H (FIG. 15D), Ab10.H (FIG. 15E), Ab21.H(FIG. 15F), Ab22 (FIG. 15G), and Ab23 (FIG. 15H) binding toPAC1-R-expressing PC-12 cells in the presence of PACAP38 obtainedfollowing the protocol in Example 6 infra. The dashed line in FIG. 15Arepresents a no PACAP and no antibody control.

FIG. 16A-H provides representative data showing Ab10-mediated (FIG.16A), Ab20-mediated (FIG. 16B), Ab21-mediated (FIG. 16C), Ab1.H-mediated(FIG. 16D), Ab10.H-mediated (FIG. 16E), Ab21.H-mediated (FIG. 16F),Ab22-mediated (FIG. 16G), and Ab23-mediated (FIG. 16H) inhibition ofPACAP38-driven cAMP production via PAC1-R-expressing PC-12 cellsobtained following the protocol in Example 1 infra.

FIG. 17A-H provides representative data showing Ab10-mediated (FIG.17A), Ab20-mediated (FIG. 17B), Ab21-mediated (FIG. 17C), Ab1.H-mediated(FIG. 17D), Ab10.H-mediated (FIG. 17E), Ab21.H-mediated (FIG. 17F),Ab22-mediated (FIG. 17G), and Ab23-mediated (FIG. 17H) inhibition ofPACAP27-driven cAMP production via PAC1-R-expressing PC-12 cellsobtained following the protocol in Example 1 infra.

FIG. 18A-H provides representative data showing Ab10-mediated (FIG.18A), Ab20-mediated (FIG. 18B), Ab21-mediated (FIG. 18C), Ab1.H-mediated(FIG. 18D), Ab10.H-mediated (FIG. 18E), Ab21.H (FIG. 18F) Ab22-mediated(FIG. 18G), and Ab23-mediated (FIG. 18H) inhibition of PACAP38-drivencAMP production via VPAC1-R-expressing CHO-K1 cells obtained followingthe protocol in Example 3 infra.

FIG. 19A-H provides representative data showing Ab10-mediated (FIG.19A), Ab20-mediated (FIG. 19B), Ab21-mediated (FIG. 19B), Ab1.H-mediated(FIG. 19D), Ab10.H-mediated (FIG. 19E), Ab21.H (FIG. 19F), Ab22-mediated(FIG. 19G), and Ab23-mediated (FIG. 19H) inhibition of PACAP38-drivencAMP production via VPAC2-R-expressing CHO-K1 cells obtained followingthe protocol in Example 4 infra.

FIG. 20 provides representative data showing a reduction in vasodilationobtained by administering Ab1.H following PACAP38 administration in arabbit model, relative to a vehicle control, obtained following theprotocol in Example 7 infra.

FIG. 21 provides representative data showing a reduction in vasodilationobtained by administering Ab10 following PACAP38 administration in arabbit model, relative to an isotype antibody control, obtainedfollowing the protocol in Example 8 infra.

FIG. 22A provides epitope binning data for labeled Ab1 and unlabeledAb10 obtained following the protocol in Example 9 infra.

FIG. 22B provides epitope binning data for unlabeled Ab1 and labeledAb10 obtained following the protocol in Example 9 infra.

FIG. 23 provides representative data showing the in vivo effect of theadministration of PACAP and an anti-PACAP antibody Ab1.H in a rodentphotophobia model, which model detects the amount of time treatedanimals (mice) spend in the light per 5 min. interval compared toappropriate control animals obtained following the protocol in Example11 infra.

FIG. 24 provides representative data showing the in vivo effect of theadministration of PACAP and anti-PACAP antibody Ab1.H in a rodentphotophobia animal model, which detects the average amount of timetreated animals (mice) spend in the light compared to appropriatecontrol animals obtained following the protocol in Example 11 infra.

FIG. 25 provides representative data showing the in vivo effect of theadministration of PACAP and an anti-PACAP antibody Ab10.H in a rodentphotophobia model, which model detects the amount of time treatedanimals (mice) spend in the light per 5 min. interval compared toappropriate control animals obtained following the protocol in Example11 infra.

FIG. 26A presents results of surface plasmon resonance-based bindingkinetics measurements for binding of anti-PACAP antibody Ab10 to PACAPalanine scanning mutants 19A, 22A, 23A, and 27A, along with controlsincluding wild-type PACAP (labelled huPACAP (1-38)) (positive control)and 1× running buffer (negative control) obtained following the protocolin Example 12 infra.

FIG. 26B presents results of surface plasmon resonance-based bindingkinetics measurements for binding of anti-PACAP antibody Ab10 to PACAPalanine scanning mutants 1A-18A, 20A, 21A, 24V-26A, and 28A-38A, alongwith controls including wild-type PACAP (labelled huPACAP (1-38))(positive control) and 1× running buffer (negative control) obtainedfollowing the protocol in Example 12 infra.

FIG. 27A presents results of surface plasmon resonance-based bindingkinetics measurements for binding of anti-PACAP antibody Ab20 to PACAPalanine scanning mutants 19A, 22A, 23A, 24V, and 27A, along withcontrols including wild-type PACAP (labelled huPACAP (1-38)) (positivecontrol) and 1× running buffer (negative control) obtained following theprotocol in Example 12 infra.

FIG. 27B presents results of surface plasmon resonance-based bindingkinetics measurements for binding of anti-PACAP antibody Ab20 to PACAPalanine scanning mutants 1A-18A, 20A, 21A, 25A, 26A, and 28A-38A, alongwith controls including wild-type PACAP (labelled huPACAP (1-38))(positive control) and 1× running buffer (negative control) obtainedfollowing the protocol in Example 12 infra.

FIG. 28A presents results of surface plasmon resonance-based bindingkinetics measurements for binding of anti-PACAP antibody Ab21 to PACAPalanine scanning mutants 19A, 22A, 23A, and 27A, along with controlsincluding wild-type PACAP (labelled huPACAP (1-38)) (positive control)and 1× running buffer (negative control) obtained following the protocolin Example 12 infra.

FIG. 28B presents results of binding kinetics measurements for bindingof anti-PACAP antibody Ab21 to PACAP alanine scanning mutants 1A-18A,20A, 21A, 24V-26A, and 28A-38A, along with controls including wild-typePACAP (labelled huPACAP (1-38)) (positive control) and 1× running buffer(negative control) obtained following the protocol in Example 12 infra.

FIG. 29A presents results of surface plasmon resonance-based bindingkinetics measurements for binding of anti-PACAP antibody Ab22 to PACAPalanine scanning mutants 22A, 23A, 27A, 28A, and 31A, along withcontrols including wild-type PACAP (labelled huPACAP (1-38)) (positivecontrol) and 1× running buffer (negative control) obtained following theprotocol in Example 12 infra.

FIG. 29B presents results of surface plasmon resonance-based bindingkinetics measurements for binding of anti-PACAP antibody Ab22 to PACAPalanine scanning mutants 1A-21A, 24V-26A, 29A, and 30A, along withcontrols including wild-type PACAP (labelled huPACAP (1-38)) (positivecontrol) and 1× running buffer (negative control) obtained following theprotocol in Example 12 infra.

FIG. 30A presents results of surface plasmon resonance-based bindingkinetics measurements for binding of anti-PACAP antibody Ab23 to PACAPalanine scanning mutants 12A, 20A, 23A, 24V, 26A, 27A, and 28A, alongwith controls including wild-type PACAP (labelled huPACAP (1-38))(positive control) and 1× running buffer (negative control) obtainedfollowing the protocol in Example 12 infra.

FIG. 30B presents results of surface plasmon resonance-based bindingkinetics measurements for binding of anti-PACAP antibody Ab23 to PACAPalanine scanning mutants 1A-11A, 13A-19A, 21A, 22A, 25V, and 29A-31A,along with controls including wild-type PACAP (labelled huPACAP (1-38))(positive control) and 1× running buffer (negative control) obtainedfollowing the protocol in Example 12 infra.

FIG. 31A presents a summary of the effects of PACAP alanine scanningmutants on antibody binding. In column 1 of FIG. 31A, VIP residues arelisted in the order of their spatial arrangement along the VIP primarysequence from amino acid residues 1-27. In column 2 of FIG. 31A, PACAPresidues are listed in the order of their spatial arrangement along thePACAP primary sequence from amino acid residues 1-27. Column 3 of FIG.31A provides the number corresponding to each residue from 1-27 for bothVIP and PACAP, as arranged spatially along their primary sequences. Incolumns 4-8 of FIG. 31A, each antibody tested during the alaninescanning studies (such as Ab10, Ab20, for example), and the PACAPresidues determined to contribute to PACAP/antibody binding, (such as5A, 6A, for example) are listed.

FIG. 31B presents a summary of the effects of PACAP alanine scanningmutants on antibody binding. In column 1 of FIG. 31B, VIP residue 28 islisted. In column 2 of FIG. 31B, PACAP residues are listed in the orderof their spatial arrangement along the PACAP primary sequence from aminoacid residues 28-38. Column 3 of FIG. 31B provides the numbercorresponding to residue 28 of VIP and each of residues 28-38 for PACAP,as arranged spatially along their primary sequences. In columns 4-8 ofFIG. 31B, each antibody tested during the alanine scanning studies (suchas Ab10, Ab20, for example), and the PACAP residues determined tocontribute to PACAP/antibody binding, (such as 5A, 6A, for example) arelisted.

DETAILED DESCRIPTION Definitions

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, animal species or genera,and reagents described, as such may vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention, which will be limited only by the appendedclaims. As used herein the singular forms “a”, “and”, and “the” includeplural referents unless the context clearly dictates otherwise. Thus,for example, reference to “a cell” includes a plurality of such cellsand reference to “the protein” includes reference to one or moreproteins and equivalents thereof known to those skilled in the art, andso forth. All technical and scientific terms used herein have the samemeaning as commonly understood to one of ordinary skill in the art towhich this invention belongs unless clearly indicated otherwise.

Pituitary Adenylate Cyclase Activating Polypeptide (PACAP):

As used herein, unless stated otherwise PACAP includes any mammalianform of PACAP, and in particular encompasses the following Homo sapiensPACAP27 and Homo sapiens PACAP38 amino acid sequences:

PACAP38:

HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNK (SEQ ID NO: 1241), wherein theC-terminal lysine is amidated; but also any mutants, splice variants,isoforms, orthologs, homologs, and variants of this sequence.

PACAP27:

HSDGIFTDSYSRYRKQMAVKKYLAAVL (SEQ ID NO: 1242), wherein the C-terminalleucine is amidated; but also any mutants, splice variants, isoforms,orthologs, homologs, and variants of this sequence.

“Photophobia” herein refers to a symptom of abnormal intolerance tovisual perception of light, sometimes additionally defined by abnormalor irrational fear of light, or by presence of actual physicalphotosensitivity of the eyes. In the present invention photophobiaincludes in particular light aversion associated with migraine, clusterheadaches and other neurological causes of light aversive behavior thatcan trigger a migraine or cluster headache. Patients/subjects candevelop photophobia as a result of several different medical conditions,related to the eye or the nervous system. Photophobia can be caused byan increased response to light starting at any step in the visual systemsuch as: (i) too much light entering the eye, (ii) too much light canenter the eye if it is damaged, such as with corneal abrasion andretinal damage, or if a pupil(s) is unable to normally constrict (seenwith damage to the oculomotor nerve), (iii) overstimulation of thephotoreceptors in the retina, (iv) excessive electric impulses to theoptic nerve, and (v) excessive response in the central nervous system.

“Effective treatment or prevention of photophobia” herein refers toinhibiting light aversive behavior or photophobia or inhibiting theonset of light aversive behavior or photophobia in a subject in needthereof, e.g., a subject having an active migraine attack or clusterheadache or a subject prone to migraine or cluster headaches, or one ofthe other photophobia-associated disorders identified herein afteradministration of an effective amount of an anti-PACAP antibody orantigen binding fragment thereof according to the invention. Thetreatment may be effected as a monotherapy or in association withanother active agent such as topiramate or dihydroergotamine by way ofexample.

The term “migraine” refers to a complex and disabling neurologicaldisorder that may progress during four stages: prodrome, aura, headache,and postdrome. A migraine is defined by the International HeadacheSociety as a headache that lasts for 4-72 hours and is characterized byat least two of the following: unilateral localization, pulsatingquality, moderate to severe pain intensity; and aggravation by movementsuch as walking. In addition, the headache must be accompanied by atleast one of the following: nausea and/or vomiting, photophobia, orphonophobia. A migraine may also be accompanied by aura, which typicallyprecedes the deadline during the premonition or prodrome phase, andoften results in visual changes, e.g., a scintillating scotoma thatmoves across the visual field. The prodrome may also be accompanied byother symptoms, e.g., fatigue, gastrointestinal issues, and moodchanges. A migraineur is often incapacitated for extended periods oftime. The postdrome is the final phase and occurs after the attack,during which time the migraineur may feel exhausted or mildly euphoric.

The term “headache” refers to pain in any region of the head. Headachesmay occur on one or both sides of the head, be isolated to a certainlocation, radiate across the head from one point, or have a vise-likequality. A headache may be a sharp pain, throbbing sensation or dullache. Headaches may appear gradually or suddenly, and they may last lessthan an hour or for several days.

The term “pain associated disease or condition” refers to any disease orcondition defined, in whole or in part, by acute and/or chronic pain.Pain is generally defined as an unpleasant sensory and emotionalexperience associated with actual or potential tissue damage, ordescribed in terms of such damage. Pain may be classified as neurogenic,neuropathic, inflammatory, or nociceptic.

The term “opioid analgesic” herein refers to all drugs, natural orsynthetic, with morphine-like actions. The synthetic and semi-syntheticopioid analgesics are derivatives of five chemical classes of compound:phenanthrenes; phenylheptylamines; phenylpiperidines; morphinans; andbenzomorphans, all of which are within the scope of the term. Exemplaryopioid analgesics include codeine, dihydrocodeine, diacetylmorphine,hydrocodone, hydromorphone, levorphanol, oxymorphone, alfentanil,buprenorphine, butorphanol, fentanyl, sufentanil, meperidine, methadone,nalbuphine, propoxyphene, and pentazocine, or pharmaceuticallyacceptable salts thereof.

The term “NSAID” refers to a non-steroidal anti-inflammatory compound.NSAIDs are categorized by virtue of their ability to inhibitcyclooxygenase. Cyclooxygenase 1 and cyclooxygenase 2 are two majorisoforms of cyclooxygenase and most standard NSAIDs are mixed inhibitorsof the two isoforms. Most standard NSAIDs fall within one of thefollowing five structural categories: (1) propionic acid derivatives,such as ibuprofen, naproxen, naprosyn, diclofenac, and ketoprofen; (2)acetic acid derivatives, such as tolmetin and sulindac; (3) fenamic acidderivatives, such as mefenamic acid and meclofenamic acid; (4)biphenylcarboxylic acid derivatives, such as diflunisal and flufenisal;and (5) oxicams, such as piroxim, sudoxicam, and isoxicam. Another classof NSAID has been described that selectively inhibit cyclooxygenase 2.COX-2 inhibitors have been described, e.g., in U.S. Pat. Nos. 5,616,601;5,604,260; 5,593,994; 5,550,142; 5,536,752; 5,521,213; 5,475,995;5,639,780; 5,604,253; 5,552,422; 5,510,368; 5,436,265; 5,409,944; and5,130,311, all of which are hereby incorporated by reference. Certainexemplary COX-2 inhibitors include celecoxib (SC-58635), DUP-697,flosulide (CGP-28238), meloxicam, 6-methoxy-2 naphthylacetic acid(6-MNA), rofecoxib, MK-966, nabumetone (prodrug for 6-MNA), nimesulide,NS-398, SC-5766, SC-58215, T-614; or combinations thereof.

As used herein, “treatment” is an approach for obtaining beneficial ordesired clinical results. For purposes of this invention, beneficial ordesired clinical results include, but are not limited to, one or more ofthe following: improvement in any aspect of PACAP-related conditionssuch as migraine or headache. For example in the context of headache ormigraine treatment this includes lessening severity, alleviation of painintensity, and other associated symptoms, reducing frequency ofrecurrence, increasing the quality of life of those suffering from theheadache, and decreasing dose of other medications required to treat theheadache. For migraine, other associated symptoms include, but are notlimited to, nausea, vomiting, and sensitivity to light, sound, and/ormovement. For cluster headache, other associated symptoms include, butare not limited to swelling under or around the eyes, excessive tears,red eye, rhinorrhea or nasal congestion, and red flushed face.

“Reducing incidence” or “prophylaxis” or “prevention” means any ofreducing severity for a particular disease, condition, symptom, ordisorder (the terms disease, condition, and disorder are usedinterchangeably throughout the application). Reduction in severityincludes reducing drugs and/or therapies generally used for thecondition by, for example, reducing the need for, amount of, and/orexposure to drugs or therapies. Reduction in severity also includesreducing the duration, and/or frequency of the particular condition,symptom, or disorder (including, for example, delaying or increasingtime to next episodic attack in an individual).

“Ameliorating” headache or one or more symptoms of headache or migraineor other PACAP-related condition means a lessening or improvement of oneor more symptoms of the condition, e.g., headache or migraine ascompared to not administering an anti-PACAP antagonist antibody.“Ameliorating” also includes shortening or reduction in duration of asymptom.

As used herein, “controlling headache” or “controlling migraine” or“controlling” another PACAP-related condition refers to maintaining orreducing severity or duration of one or more symptoms of the condition,e.g., headache or migraine or frequency of headache or migraine attacksin an individual (as compared to the level before treatment). Forexample, the duration or severity of head pain, or frequency of attacksis reduced by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, or 100% in the individual as compared to the level beforetreatment. The reduction in the duration or severity of head pain, orfrequency of attacks can last for any length of time, e.g., 2 weeks, 4weeks (1 month), 8 weeks (2 months), 16 weeks (3 months), 4 months, 5months, 6 months, 9 months, 12 months, etc.

As used therein, “delaying” the development of a PACAP-related conditionsuch as migraine or headache means to defer, hinder, slow, retard,stabilize, and/or postpone progression of the condition or disease. Thisdelay can be of varying lengths of time, depending on the history of thecondition or disease and/or individuals being treated. As is evident toone skilled in the art, a sufficient or significant delay can, ineffect, encompass prevention, in that the individual does not developheadache (e.g., migraine). A method that “delays” development of thesymptom is a method that reduces probability of developing the symptomin a given time frame and/or reduces extent of the symptoms in a giventime frame, when compared to not using the method. Such comparisons aretypically based on clinical studies, using a statistically significantnumber of subjects.

“Development” or “progression” of a PACAP-related condition such asmigraine or headache means initial manifestations and/or ensuingprogression of the disorder. Development of headache or migraine can bedetectable and assessed using standard clinical techniques as well knownin the art. However, development also refers to progression that may beundetectable. For purpose of this invention, development, or progressionrefers to the biological course of the symptoms. “Development” includesoccurrence, recurrence, and onset. As used herein “onset” or“occurrence” of a condition such as headache or migraine includesinitial onset and/or recurrence.

As used herein, an “effective dosage” or “effective amount” of drug,compound, or pharmaceutical composition is an amount sufficient toeffect beneficial or desired results. For prophylactic use, beneficialor desired results include results such as eliminating or reducing therisk, lessening the severity, or delaying the outset of the disease,including biochemical, histological, and/or behavioral symptoms of thedisease, its complications and intermediate pathological phenotypespresenting during development of the disease. For therapeutic use,beneficial or desired results include clinical results such as reducingpain intensity, duration, or frequency of headache attack, anddecreasing one or more symptoms resulting from headache (biochemical,histological, and/or behavioral), including its complications andintermediate pathological phenotypes presenting during development ofthe disease, increasing the quality of life of those suffering from thedisease, decreasing the dose of other medications required to treat thedisease, enhancing effect of another medication, and/or delaying theprogression of the disease of patients. An effective dosage can beadministered in one or more administrations. For purposes of thisinvention, an effective dosage of drug, compound, or pharmaceuticalcomposition is an amount sufficient to accomplish prophylactic ortherapeutic treatment either directly or indirectly. As is understood inthe clinical context, an effective dosage of a drug, compound, orpharmaceutical composition may or may not be achieved in conjunctionwith another drug, compound, or pharmaceutical composition. Thus, an“effective dosage” may be considered in the context of administering oneor more therapeutic agents, and a single agent may be considered to begiven in an effective amount if, in conjunction with one or more otheragents, a desirable result may be or is achieved.

A “suitable host cell” or “host cell” generally includes any cellwherein the subject anti-PACAP antibodies and antigen binding fragmentsthereof can be produced recombinantly using techniques and materialsreadily available. For example, the anti-PACAP antibodies and antigenbinding fragments thereof of the present invention can be produced ingenetically engineered host cells according to conventional techniques.Suitable host cells are those cell types that can be transformed ortransfected with exogenous DNA and grown in culture, and includebacteria, fungal cells (e.g., yeast), and cultured higher eukaryoticcells (including cultured cells of multicellular organisms),particularly cultured mammalian cells, e.g., human or non-humanmammalian cells. In an exemplary embodiment these antibodies may beexpressed in CHO cells. Techniques for manipulating cloned DNA moleculesand introducing exogenous DNA into a variety of host cells are disclosedby Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed.,Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press (1989),and Current Protocols in Molecular Biology, Ausubel et al., editors, NewYork, N.Y.: Green and Wiley and Sons (1993).

In some exemplary embodiments the antibodies may be expressed in matingcompetent yeast, e.g., any haploid, diploid, or tetraploid yeast thatcan be grown in culture. Yeast useful in fermentation expression methodsmay exist in a haploid, diploid, or other polyploid form. The cells of agiven ploidy may, under appropriate conditions, proliferate for anindefinite number of generations in that form. Diploid cells can alsosporulate to form haploid cells. Sequential mating can result intetraploid strains through further mating or fusion of diploid strains.The present invention contemplates the use of haploid yeast, as well asdiploid or other polyploid yeast cells produced, for example, by matingor spheroplast fusion. By way of example, such yeast may include membersof the Saccharomycetaceae family, which includes the genera Arxiozyma;Ascobotryozyma; Citeromyces; Debaryomyces; Dekkera; Eremothecium;Issatchenkia; Kazachstania; Kluyveromyces; Kodamaea; Lodderomyces;Pachysolen; Pichia; Saccharomyces; Saturnispora; Tetrapisispora;Torulaspora; Williopsis; and Zygosaccharomyces. Other types of yeastpotentially useful in the invention include Yarrowia; Rhodosporidium;Candida; Hansenula; Filobasium; Sporidiobolus; Bullera; Leucosporidiumand Filobasidella.

In a preferred exemplary embodiment of the invention, the matingcompetent yeast used for antibody expression may comprise a member ofthe genus Pichia. In a further preferred exemplary embodiment of theinvention, the mating competent yeast of the genus Pichia is one of thefollowing species: Pichia pastoris, Pichia methanolica, and Hansenulapolymorpha (Pichia angusta). In a particularly preferred embodiment ofthe invention, the mating competent yeast of the genus Pichia is thespecies Pichia pastoris.

A “selectable marker” herein refers to a gene or gene fragment thatconfers a growth phenotype (physical growth characteristic) on a cellreceiving that gene as, for example through a transformation event. Theselectable marker allows that cell to survive and grow in a selectivegrowth medium under conditions in which cells that do not receive thatselectable marker gene cannot grow. Selectable marker genes generallyfall into several types, including positive selectable marker genes suchas a gene that confers on a cell resistance to an antibiotic or otherdrug, temperature when two temperature sensitive (“ts”) mutants arecrossed or a is mutant is transformed; negative selectable marker genessuch as a biosynthetic gene that confers on a cell the ability to growin a medium without a specific nutrient needed by all cells that do nothave that biosynthetic gene, or a mutagenized biosynthetic gene thatconfers on a cell inability to grow by cells that do not have the wildtype gene; and the like. Suitable markers include but are not limitedto: ZEO; G418; LYS3; MET1; MET3a; ADE1; ADE3; URA3; and the like.

An “expression vector” herein refers to DNA vectors containing elementsthat facilitate manipulation for the expression of a foreign proteinwithin the target host cell, e.g., a bacterial, insect, yeast, plant,amphibian, reptile, avian, or mammalian cell, and most typically a yeastor mammalian cell, e.g., a CHO cell. Conveniently, manipulation ofsequences and production of DNA for transformation is first performed ina bacterial host, e.g. E. coli, and usually vectors will includesequences to facilitate such manipulations, including a bacterial originof replication and appropriate bacterial selection marker. Selectionmarkers encode proteins necessary for the survival or growth oftransformed host cells grown in a selective culture medium. Host cellsnot transformed with the vector containing the selection gene will notsurvive in the culture medium. Typical selection genes encode proteinsthat (a) confer resistance to antibiotics or other toxins, (b)complement auxotrophic deficiencies, or (c) supply critical nutrientsnot available from complex media. Exemplary vectors and methods fortransformation of yeast are described, for example, in Burke, D.,Dawson, D., & Stearns, T., Methods in yeast genetics: a Cold SpringHarbor Laboratory course manual, Plainview, N.Y.: Cold Spring HarborLaboratory Press (2000).

Expression vectors for use in the methods of the invention may includeyeast or mammalian specific sequences, including a selectableauxotrophic or drug marker for identifying transformed host strains. Adrug marker may further be used to amplify copy number of the vector ina yeast host cell.

The polypeptide coding sequence of interest is operably linked totranscriptional and translational regulatory sequences that provide forexpression of the polypeptide in the desired host cells, e.g., yeast ormammalian cells. These vector components may include, but are notlimited to, one or more of the following: an enhancer element, apromoter, and a transcription termination sequence. Sequences for thesecretion of the polypeptide may also be included, e.g. a signalsequence, and the like. An origin of replication, e.g., a yeast originof replication, is optional, as expression vectors are often integratedinto the host cell genome. In one embodiment of the invention, thepolypeptide of interest is operably linked, or fused, to sequencesproviding for optimized secretion of the polypeptide from yeast diploidcells.

Nucleic acids are “operably linked” when placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for asignal sequence is operably linked to DNA for a polypeptide if it isexpressed as a preprotein that participates in the secretion of thepolypeptide; a promoter or enhancer is operably linked to a codingsequence if it affects the transcription of the sequence. Generally,“operably linked” means that the DNA sequences being linked arecontiguous, and, in the case of a secretory leader, contiguous and inreading frame. However, enhancers do not have to be contiguous. Linkingis accomplished by ligation at convenient restriction sites oralternatively via a PCR/recombination method familiar to those skilledin the art (GATEWAY® Technology; Invitrogen, Carlsbad Calif.). If suchsites do not exist, the synthetic oligonucleotide adapters or linkersare used in accordance with conventional practice.

Promoters are untranslated sequences located upstream (5′) to the startcodon of a structural gene (generally within about 100 to 1000 bp) thatcontrol the transcription and translation of particular nucleic acidsequences to which they are operably linked. Such promoters fall intoseveral classes: inducible, constitutive, and repressible promoters(that increase levels of transcription in response to absence of arepressor). Inducible promoters may initiate increased levels oftranscription from DNA under their control in response to some change inculture conditions, e.g., the presence or absence of a nutrient or achange in temperature.

The promoter fragment may also serve as the site for homologousrecombination and integration of the expression vector into the samesite in the host cell, e.g., yeast cell, genome; alternatively, aselectable marker may be used as the site for homologous recombination.Pichia transformation is described in Cregg et al., Mol. Cell. Biol.,5:3376-3385 (1985). Suitable promoters for use in different eukaryoticand prokaryotic cells are well known and commercially available.

The polypeptides of interest may be produced recombinantly not onlydirectly, but also as a fusion polypeptide with a heterologouspolypeptide, e.g. a signal sequence or other polypeptide having aspecific cleavage site at the N-terminus of the mature protein orpolypeptide. In general, the signal sequence may be a component of thevector, or it may be a part of the polypeptide coding sequence that isinserted into the vector. The heterologous signal sequence selectedpreferably is one that is recognized and processed through one of thestandard pathways available within the host cell, e.g., a mammaliancell, an insect cell, or a yeast cell. Additionally, these signalpeptide sequences may be engineered to provide for enhanced secretion inexpression systems. Secretion signals of interest also include mammalianand yeast signal sequences, which may be heterologous to the proteinbeing secreted, or may be a native sequence for the protein beingsecreted. Signal sequences include pre-peptide sequences, and in someinstances may include propeptide sequences. Many such signal sequencesare known in the art, including the signal sequences found onimmunoglobulin chains, e.g., K28 preprotoxin sequence, PHA-E, FACE,human MCP-1, human serum albumin signal sequences, human Ig heavy chain,human Ig light chain, and the like. For example, see Hashimoto et. al.,Protein Eng., 11(2):75 (1998); and Kobayashi et. al., TherapeuticApheresis, 2(4):257 (1998).

Transcription may be increased by inserting a transcriptional activatorsequence into the vector. These activators are cis-acting elements ofDNA, usually about from 10 to 300 bp, which act on a promoter toincrease its transcription. Transcriptional enhancers are relativelyorientation and position independent, having been found 5′ and 3′ to thetranscription unit, within an intron, as well as within the codingsequence itself. The enhancer may be spliced into the expression vectorat a position 5′ or 3′ to the coding sequence, but is preferably locatedat a site 5′ from the promoter.

Expression vectors used in eukaryotic host cells may also containsequences necessary for the termination of transcription and forstabilizing the mRNA. Such sequences are commonly available from 3′ tothe translation termination codon, in untranslated regions of eukaryoticor viral DNAs or cDNAs. These regions contain nucleotide segmentstranscribed as polyadenylated fragments in the untranslated portion ofthe mRNA.

Construction of suitable vectors containing one or more of theabove-listed components employs standard ligation techniques orPCR/recombination methods. Isolated plasmids or DNA fragments arecleaved, tailored, and re-ligated in the form desired to generate theplasmids required or via recombination methods. For analysis to confirmcorrect sequences in plasmids constructed, the ligation mixtures areused to transform host cells, and successful transformants selected byantibiotic resistance (e.g. ampicillin or Zeocin) where appropriate.Plasmids from the transformants are prepared, analyzed by restrictionendonuclease digestion, and/or sequenced.

As an alternative to restriction and ligation of fragments,recombination methods based on specific attachment (“att”) sites andrecombination enzymes may be used to insert DNA sequences into a vector.Such methods are described, for example, by Landy, Ann. Rev. Biochem.,58:913-949 (1989); and are known to those of skill in the art. Suchmethods utilize intermolecular DNA recombination that is mediated by amixture of lambda and E. coli—encoded recombination proteins.Recombination occurs between att sites on the interacting DNA molecules.For a description of att sites see Weisberg and Landy, Site-SpecificRecombination in Phage Lambda, in Lambda II, p. 211-250, Cold SpringHarbor, N.Y.: Cold Spring Harbor Press (1983). The DNA segments flankingthe recombination sites are switched, such that after recombination, theatt sites are hybrid sequences comprised of sequences donated by eachparental vector. The recombination can occur between DNAs of anytopology.

Att sites may be introduced into a sequence of interest by ligating thesequence of interest into an appropriate vector; generating a PCRproduct containing att B sites through the use of specific primers;generating a cDNA library cloned into an appropriate vector containingatt sites; and the like.

Folding, as used herein, refers to the three-dimensional structure ofpolypeptides and proteins, where interactions between amino acidresidues act to stabilize the structure. While non-covalent interactionsare important in determining structure, usually the proteins of interestwill have intra- and/or intermolecular covalent disulfide bonds formedby two cysteine residues. For naturally occurring proteins andpolypeptides or derivatives and variants thereof, the proper folding istypically the arrangement that results in optimal biological activity,and can conveniently be monitored by assays for activity, e.g. ligandbinding, enzymatic activity, etc.

In some instances, for example where the desired product is of syntheticorigin, assays based on biological activity will be less meaningful. Theproper folding of such molecules may be determined on the basis ofphysical properties, energetic considerations, modeling studies, and thelike.

The expression host may be further modified by the introduction ofsequences encoding one or more enzymes that enhance folding anddisulfide bond formation, i.e. foldases, chaperonins, etc. Suchsequences may be constitutively or inducibly expressed in the yeast hostcell, using vectors, markers, etc. as known in the art. Preferably thesequences, including transcriptional regulatory elements sufficient forthe desired pattern of expression, are stably integrated in the yeastgenome through a targeted methodology.

For example, the eukaryotic protein disulfide isomerase (“PDI”) is notonly an efficient catalyst of protein cysteine oxidation and disulfidebond isomerization, but also exhibits chaperone activity. Co-expressionof PDI can facilitate the production of active proteins having multipledisulfide bonds. Also of interest is the expression of immunoglobulinheavy chain binding protein (“BIP”); cyclophilin; and the like. In oneembodiment of the invention, each of the haploid parental strainsexpresses a distinct folding enzyme, e.g. one strain may express BIP,and the other strain may express PDI or combinations thereof.

Cultured mammalian cells are also preferred exemplary hosts forproduction of the disclosed anti-PACAP antibodies and antigen bindingfragments thereof. As mentioned, CHO cells are particularly suitable forexpression of antibodies. Many procedures are known in the art formanufacturing monoclonal antibodies in mammalian cells. (See, Galfre, G.and Milstein, C., Methods Enzym., 73:3-46, 1981; Basalp et al., Turk. J.Biol., 24:189-196, 2000; Wurm, F. M., Nat. Biotechnol., 22:1393-1398,2004; and Li et al., mAbs, 2(5):466-477, 2010). As mentioned in furtherdetail infra, common host cell lines employed in mammalian monoclonalantibody manufacturing schemes include, but are not limited to, humanembryonic retinoblast cell line PER.C6® (Crucell N. V., Leiden, TheNetherlands), NS0 murine myeloma cells (Medical Research Council,London, UK), CV1 monkey kidney cell line, 293 human embryonic kidneycell line, BHK baby hamster kidney cell line, VERO African green monkeykidney cell line, human cervical carcinoma cell line HELA, MDCK caninekidney cells, BRL buffalo rat liver cells, W138 human lung cells, HepG2human liver cells, MMT mouse mammary tumor cells, TRI cells, MRCS cells,F_(s)4 cells, myeloma or lymphoma cells, or Chinese Hamster (Cricetulusgriseus) Ovary (CHO) cells, and the like. Many different subclones orsub-cell lines of CHO cells known in the art that are useful andoptimized for production of recombinant monoclonal antibodies, such asthe DP12 (CHO K1 dhfr−) cell line, NS0 cells are a non-Ig secreting,non-light chain-synthesizing subclone of NS-1 cells that are resistantto azaguanine. Other Chinese Hamster and CHO cells are commerciallyavailable (from ATCC, etc.), including CHO-DXB 11 (CHO-DUKX), CHO-pro3,CHO-DG44, CHO 1-15, CHO DP-12, Lec2, M1WT3, Lec8, pgsA-745, and thelike, all of which are genetically altered to optimize the cell line forvarious parameters. Monoclonal antibodies are commonly manufacturedusing a batch fed method whereby the monoclonal antibody chains areexpressed in a mammalian cell line and secreted into the tissue culturemedium in a bioreactor. Medium (or feed) is continuously supplied to thebioreactor to maximize recombinant protein expression. Recombinantmonoclonal antibody is then purified from the collected media. In somecircumstances, additional steps are needed to reassemble the antibodiesthrough reduction of disulfide bonds, etc. Such production methods canbe scaled to be as large as 10,000 L in a single batch or more. It isnow routine to obtain as much as 20 pg/cell/day through the use of suchcell lines and methodologies, providing titers as high as 10 g/L ormore, amounting to 15 to 100 kg from bioreactors of 10 kL to 25 kL. (Liet al., 2010). Various details of this production methodology, includingcloning of the polynucleotides encoding the antibodies into expressionvectors, transfecting cells with these expression vectors, selecting fortransfected cells, and expressing and purifying the recombinantmonoclonal antibodies from these cells are provided below.

For recombinant production of an anti-PACAP antibody or antigen bindingfragment in mammalian cells, nucleic acids encoding the antibody orfragment thereof are generally inserted into a replicable vector forfurther cloning (amplification of the DNA) or for expression. DNAencoding the antibody is readily isolated or synthesized usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to DNAs encoding the heavy and lightchains of the antibody). The vector components generally include, butare not limited to, one or more of the following: a signal sequence, anorigin of replication, one or more marker genes, an enhancer element, apromoter, and a transcription termination sequence. Selection ofpromoters, terminators, selectable markers, vectors, and other elementsis a matter of routine design within the level of ordinary skill in theart. Many such elements are known in the art and are available throughcommercial suppliers.

The antibodies of this invention may be produced recombinantly not onlydirectly, but also as a fusion polypeptide with a heterologouspolypeptide, which is preferably a signal sequence or other polypeptidehaving a specific cleavage site at the N-terminus of the mature proteinor polypeptide. The homologous or heterologous signal sequence selectedpreferably is one that is recognized and processed (i.e., cleaved by asignal peptidase) by the host cell. In mammalian cell expression,mammalian signal sequences as well as viral secretory leaders, forexample, the herpes simplex gD signal, are available.

Such expression vectors and cloning vectors will generally contain anucleic acid sequence that enables the vector to replicate in one ormore selected host cells. Typically, in cloning vectors this sequence isone that enables the vector to replicate independently of the hostchromosomal DNA, and includes origins of replication or autonomouslyreplicating sequences. Such sequences are well known for a variety ofbacteria, yeast, and viruses, e.g., the origin of replication from theplasmid pBR322 is suitable for most Gram-negative bacteria, the 2muplasmid origin is suitable for yeast, and various viral origins (SimianVirus 40 (“SV40”), polyoma, adenovirus, vesicular stomatitis virus(“VSV”), or bovine papillomavirus (“BPV”) are useful for cloning vectorsin mammalian cells. Generally, the origin of replication component isnot needed for mammalian expression vectors (the SV40 origin maytypically be used only because it contains the early promoter).

These vectors will also typically contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Drug selection is generally used to select for culturedmammalian cells into which foreign DNA has been inserted. Such cells arecommonly referred to as “transfectants”. Cells that have been culturedin the presence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.”Examples of such dominant selection use the drugs neomycin, mycophenolicacid, and hygromycin. An exemplary selectable marker is a gene encodingresistance to the antibiotic neomycin. Selection is carried out in thepresence of a neomycin-type drug, such as G-418 or the like. Those cellsthat are successfully transformed with a heterologous gene produce aprotein conferring drug resistance and thus survive the selectionregimen.

Selection systems can also be used to increase the expression level ofthe gene of interest, a process referred to as “amplification.”Amplification of transfectants typically occurs by culturing the cellsin the presence of a low level of the selective agent and thenincreasing the amount of selective agent to select for cells thatproduce high levels of the products of the introduced genes. Exemplarysuitable selectable markers for mammalian cells are those that enablethe identification of cells competent to take up the antibody nucleicacid, such as dihydrofolate reductase (“DHFR”), thymidine kinase,metallothionein-I and -II, preferably primate metallothionein genes,adenosine deaminase, ornithine decarboxylase, etc.

For example, an amplifiable selectable marker for mammalian cells isdihydrofolate reductase, which confers resistance to methotrexate. Otherdrug resistance genes (e.g. hygromycin resistance, multi-drugresistance, puromycin acetyltransferase) can also be used. Cellstransformed with the DHFR selection gene are first identified byculturing all of the transformants in a culture medium that containsmethotrexate (“MTX”), a competitive antagonist of DHFR. An appropriatehost cell when wild-type DHFR is employed is the Chinese hamster ovary(“CHO”) cell line deficient in DHFR activity.

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding antibody, wild-type DHFR protein, and another selectable markersuch as aminoglycoside 3′-phosphotransferase (“APH”) can be selected bycell growth in medium containing a selection agent for the selectablemarker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin,or G-418. See U.S. Pat. No. 4,965,199.

These vectors may comprise an enhancer sequence that facilitatestranscription of a DNA encoding the antibody. Many enhancer sequencesare known from mammalian genes (for example, globin, elastase, albumin,alpha-fetoprotein, and insulin). A frequently used enhancer is onederived from a eukaryotic cell virus. Examples thereof include the SV40enhancer on the late side of the replication origin (bp 100-270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers (See,Yaniv, Nature, 297:17-18, 1982, on enhancing elements for activation ofeukaryotic promoters). The enhancer may be spliced into the vector at aposition 5′ or 3′ to the antibody-encoding sequence, but is preferablylocated at a site 5′ from the promoter.

Expression and cloning vectors will also generally comprise a promoterthat is recognized by the host organism and is operably linked to theantibody nucleic acid. Promoter sequences are known for eukaryotes.Virtually all eukaryotic genes have an AT-rich region locatedapproximately 25 to 30 bases upstream from the site where transcriptionis initiated. Another sequence found 70 to 80 bases upstream from thestart of transcription of many genes is a CNCAAT region where N may beany nucleotide. At the 3′ end of most eukaryotic genes is an AATAAAsequence that may be the signal for addition of the poly A tail to the3′ end of the coding sequence. All of these sequences are suitablyinserted into eukaryotic expression vectors.

Antibody transcription from vectors in mammalian host cells iscontrolled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), BPV, avian sarcoma virus, cytomegalovirus, a retrovirus,hepatitis-B virus, and most preferably SV40, from heterologous mammalianpromoters, e.g., the actin promoter or an immunoglobulin promoter, fromheat-shock promoters, provided such promoters are compatible with thehost cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using the BPVas a vector is disclosed in U.S. Pat. No. 4,419,446. A modification ofthis system is described in U.S. Pat. No. 4,601,978. See also Reyes etal., Nature, 297:598-601 (1982) on expression of human beta-interferoncDNA in mouse cells under the control of a thymidine kinase promoterfrom herpes simplex virus. Alternatively, the rous sarcoma virus longterminal repeat can be used as the promoter.

Strong transcription promoters can be used, such as promoters from SV40,cytomegalovirus, or myeloproliferative sarcoma virus. See, e.g., U.S.Pat. No. 4,956,288 and U.S. Patent Publication No. 20030103986. Othersuitable promoters include those from metallothionein genes (U.S. Pat.Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.Expression vectors for use in mammalian cells include pZP-1, pZP-9, andpZMP21, which have been deposited with the American Type CultureCollection, 10801 University Blvd., Manassas, Va. USA under accessionnumbers 98669, 98668, and PTA-5266, respectively, and derivatives ofthese vectors.

Expression vectors used in eukaryotic host cells (yeast, fungus, insect,plant, animal, human, or a nucleated cell from other multicellularorganism) will also generally contain sequences necessary for thetermination of transcription and for stabilizing the mRNA. Suchsequences are commonly available from the 5′ and, occasionally 3′,untranslated regions of eukaryotic or viral DNAs or cDNAs. These regionscontain nucleotide segments transcribed as polyadenylated fragments inthe untranslated portion of the mRNA encoding the antibody. One usefultranscription termination component is the bovine growth hormonepolyadenylation region. See WO 94/11026 and the expression vectordisclosed therein.

Suitable host cells for cloning or expressing the subject antibodiesinclude prokaryote, yeast, or higher eukaryote cells described above.However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture has become a routine procedure. Examplesof useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-1 (ATCC No. CRL 1650); and COS-7, ATCC CRL1651); human embryonic kidney line (293 or 293 cells subcloned forgrowth in suspension culture, (ATCC No. CRL 1573; Graham et al., J. Gen.Virol., 36:59-72 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10,ATCC No. CRL 1632; BHK 570, ATCC No. CRL 10314); CHO cells (CHO-K1, ATCCNo. CCL 61; CHO-DG44, Urlaub et al., Proc. Natl. Acad. Sci. USA,77:4216-4220 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.,23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad.Sci., 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatomaline (Hep G2). Additional suitable cell lines are known in the art andavailable from public depositories such as the American Type CultureCollection, Manassas, Va.

Host cells are transformed with the above-described expression orcloning vectors for antibody production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences asdiscussed supra.

The mammalian host cells used to produce the antibody of this inventionmay be cultured in a variety of media. Commercially available media suchas Ham's F10 (Sigma-Aldrich Corporation, St. Louis, Mo.), MinimalEssential Medium ((“MEM” (Sigma-Aldrich Corporation, St. Louis, Mo.),Roswell Park Memorial Institute-1640 medium (“RPMI-1640”, Sigma-AldrichCorporation, St. Louis, Mo.), and Dulbecco's Modified Eagle's Medium((“DMEM” Sigma-Aldrich Corporation, St. Louis, Mo.) are suitable forculturing the host cells. In addition, any of the media described in Hamet al., Meth. Enz., 58:44 (1979); Barnes et al., Anal. Biochem., 102:255(1980); U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Reexam No. 30,985 canbe used as culture media for the host cells. Any of these media may besupplemented as necessary with hormones and/or other growth factors(such as insulin, transferrin, or epidermal growth factor), salts (suchas sodium chloride, calcium, magnesium, and phosphate), buffers (such asHEPES), nucleotides (such as adenosine and thymidine), antibiotics (suchas Gentamycin drug), trace elements (defined as inorganic compoundsusually present at final concentrations in the micromolar range), andglucose or an equivalent energy source. Any other necessary supplementsmay also be included at appropriate concentrations that would be knownto those skilled in the art. The culture conditions, such astemperature, pH, and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan. Methods of development and optimization of media andculture conditions are known in the art. (See, Gronemeyer et al.,Bioengineering, 1(4): 188-212, 2014).

After culture conditions are optimized and a preferred cell line cloneis selected, these cells are cultured (either adherent cells orsuspension cultures) most typically in a batch-fed process in abioreactor (many models are commercially available) that involvescontinuously feeding the cell culture with medium and feed, optimizedfor the particular cell line chosen and selected for this purpose. (See,Butler, M., Appl. Microbiol. Biotechnol., 68:283-291, 2005; and Kelley,B., mAb, 1(5):443-452, 2009). Perfusion systems are also available inwhich media and feed are continuously supplied to the culture while thesame volume of media is being withdrawn from the bioreactor. (Wurm,2004). Synthetic media, also commercially available, are available forgrowing cells in a batch-fed culture, avoiding the possibility ofcontamination from outside sources, such as with the use of animalcomponents, such as bovine serum albumin, etc. However,animal-component-free hydrolysates are commercially available to helpboost cell density, culture viability and productivity. (Li et al.,2010). Many studies have been performed in an effort to optimize cellculture media, including careful attention to head space available inroller bottles, redox potentials during growth and expression phases,presence of reducing agents to maintain disulfide bonds duringproduction, etc. (See, for instance, Hutterer et al., mAbs,5(4):608-613, 2013; and Mullan et al., BMC Proceed., 5(Suppl 8):P110,2011). Various methodologies have been developed to address thepossibility of harmful oxidation during recombinant monoclonal antibodyproduction. (See, for example, U.S. Pat. No. 8,574,869). Cultured cellsmay be grown by feeding nutrients continuously or as separatelyadministered amounts. Often various process parameters such as cellconcentration, pH, temperature, CO₂, dO₂, osmolality, amount ofmetabolites such as glucose, lactate, glutamine and glutamate, and thelike, are monitored by the use of probes during the cell growth eitheron-line by direct connection to calibrated analyzers or off-line byintervention of operators. The culturing step also typically involvesensuring that the cells growing in culture maintain the transfectedrecombinant genes by any means known in the art for cell selection.

Following fermentation, i.e., upon reaching maximum cell growth andrecombinant protein expression, the culturing step is typically followedby a harvesting step, whereby the cells are separated from the mediumand a harvested cell culture media is thereby obtained. (See, Liu etal., mAbs, 2(5):480-499, 2010). Typically various purification steps,involving column chromatography and the like, follow culturing toseparate the recombinant monoclonal antibody from cell components andcell culture media components. The exact purification steps needed forthis phase of the production of recombinant monoclonal antibodiesdepends on the site of expression of the proteins, i.e., in the cytosolof the cells themselves, or the more commonly preferred route of proteinexcreted into the cell culture medium. Various cell components may beseparated using techniques known in the art such as differentialcentrifugation techniques, gravity-based cell settling, and/or sizeexclusion chromatograph/filtration techniques that can includetangential flow micro-filtration or depth filtration. (See, Pollock etal., Biotechnol. Bioeng., 110:206-219, 2013, and Liu et al., 2010).Centrifugation of cell components may be achieved on a large scale byuse of continuous disk stack centrifuges followed by clarification usingdepth and membrane filters. (See, Kelley, 2009). Most often, afterclarification, the recombinant protein is further purified by Protein Achromatography due to the high affinity of Protein A for the Fc domainof antibodies, and typically occurs using a low pH/acidification elutionstep (typically the acidification step is combined with a precautionaryvirus inactivation step). Flocculation and/or precipitation steps usingacidic or cationic polyelectrolytes may also be employed to separateanimal cells in suspension cultures from soluble proteins. (Liu et al.,2010). Lastly, anion- and cation-exchange chromatography, hydrophobicinteraction chromatograph (“HIC”), hydrophobic charge inductionchromatograph (HCIC), hydroxyapatite chromatography using ceramichydroxyapatite (Ca₅(PO₄)₃OH)₂, and combinations of these techniques aretypically used to polish the solution of recombinant monoclonalantibody. Final formulation and concentration of the desired monoclonalantibody may be achieved by use of ultracentrifugation techniques.Purification yields are typically 70 to 80%. (Kelley, 2009).

The terms “desired protein” or “desired antibody” are usedinterchangeably and refer generally to a parent antibody specific to atarget, i.e., PACAP or a chimeric or humanized antibody or a bindingportion thereof derived therefrom as described herein. The term“antibody” is intended to include any polypeptide chain-containingmolecular structure with a specific shape that fits to and recognizes anepitope, where one or more non-covalent binding interactions stabilizethe complex between the molecular structure and the epitope. Thearchetypal antibody molecule is the immunoglobulin, and all types ofimmunoglobulins, IgG, IgM, IgA, IgE, IgD, etc., from all sources, e.g.human, rodent, rabbit, cow, sheep, pig, dog, other mammals, chicken,other avians, etc., are considered to be “antibodies.” A preferredsource for producing antibodies useful as starting material according tothe invention is rabbits. Examples thereof include chimeric antibodies,human antibodies and other non-human mammalian antibodies, humanizedantibodies, single chain antibodies (such as scFvs), camelbodies,nanobodies, IgNAR (single-chain antibodies which may be derived fromsharks, for example), small-modular immunopharmaceuticals (“SMIPs”), andantibody fragments such as Fabs, Fab′, F(ab′)₂, and the like (See,Streltsov et al., Protein Sci., 14(11):2901-9, 2005; Greenberg et al.,Nature, 374(6518):168-73, 1995; Nuttall et al., Mol. Immunol.,38(4):313-26, 2001; Hamers-Casterman et al., Nature, 363(6428):446-8,1993; Gill et al., Curr. Opin. Biotechnol., (6):653-8, 2006).

For example, antibodies or antigen binding fragments thereof may beproduced by genetic engineering. In this technique, as with othermethods, antibody-producing cells are sensitized to the desired antigenor immunogen. The messenger RNA isolated from antibody producing cellsis used as a template to make cDNA using PCR amplification. A library ofvectors, each containing one heavy chain gene and one light chain generetaining the initial antigen specificity, is produced by insertion ofappropriate sections of the amplified immunoglobulin cDNA into theexpression vectors. A combinatorial library is constructed by combiningthe heavy chain gene library with the light chain gene library. Thisresults in a library of clones that co-express a heavy and light chain(resembling the Fab fragment or antigen binding fragment of an antibodymolecule). The vectors that carry these genes are co-transfected into ahost cell. When antibody gene synthesis is induced in the transfectedhost, the heavy and light chain proteins self-assemble to produce activeantibodies that can be detected by screening with the antigen orimmunogen.

Antibody coding sequences of interest include those encoded by nativesequences, as well as nucleic acids that, by virtue of the degeneracy ofthe genetic code, are not identical in sequence to the disclosed nucleicacids, and variants thereof. Variant polypeptides can include amino acid(“aa”) substitutions, additions, or deletions. The amino acidsubstitutions can be conservative amino acid substitutions orsubstitutions to eliminate non-essential amino acids, such as to alter aglycosylation site, or to minimize misfolding by substitution ordeletion of one or more cysteine residues that are not necessary forfunction. Variants can be designed so as to retain or have enhancedbiological activity of a particular region of the protein (e.g., afunctional domain, catalytic amino acid residues, etc.). Variants alsoinclude fragments of the polypeptides disclosed herein, particularlybiologically active fragments and/or fragments corresponding tofunctional domains. Techniques for in vitro mutagenesis of cloned genesare known. Also included in the subject invention are polypeptides thathave been modified using ordinary molecular biological techniques so asto improve their resistance to proteolytic degradation or to optimizesolubility properties or to render them more suitable as a therapeuticagent.

Chimeric antibodies may be made by recombinant means by combining theV_(L) and V_(H) regions, obtained from antibody producing cells of onespecies with the constant light and heavy chain regions from another.Typically chimeric antibodies utilize rodent or rabbit variable regionsand human constant regions, in order to produce an antibody withpredominantly human domains. The production of such chimeric antibodiesis well known in the art, and may be achieved by standard means (asdescribed, e.g., in U.S. Pat. No. 5,624,659, incorporated herein byreference in its entirety). It is further contemplated that the humanconstant regions of chimeric antibodies of the invention may be selectedfrom IgG1, IgG2, IgG3, and IgG4 constant regions.

Humanized antibodies are engineered to contain even more human-likeimmunoglobulin domains, and incorporate only the complementaritydetermining regions of the animal-derived antibody. This is accomplishedby carefully examining the sequence of the hyper-variable loops of thevariable regions of the monoclonal antibody, and fitting them to thestructure of the human antibody chains. Although facially complex, theprocess is straightforward in practice. See, e.g., U.S. Pat. No.6,187,287, incorporated fully herein by reference.

In addition to entire immunoglobulins (or their recombinantcounterparts), immunoglobulin fragments comprising the epitope bindingsite (e.g., Fab′, F(ab′)₂, or other fragments) may be synthesized.“Fragment” or minimal immunoglobulins may be designed utilizingrecombinant immunoglobulin techniques. For instance “Fv” immunoglobulinsfor use in the present invention may be produced by synthesizing a fusedvariable light chain region and a variable heavy chain region.Combinations of antibodies are also of interest, e.g. diabodies, whichcomprise two distinct Fv specificities. In another embodiment of theinvention, small molecule immunopharmaceuticals (“SMIPs”), camelbodies,nanobodies, and IgNAR are encompassed by immunoglobulin fragments.

Immunoglobulins and fragments thereof may be modifiedpost-translationally, e.g. to add effector moieties such as chemicallinkers, detectable moieties, such as fluorescent dyes, enzymes, toxins,substrates, bioluminescent materials, radioactive materials,chemiluminescent moieties, and the like, or specific binding moieties,such as streptavidin, avidin, or biotin, and the like may be utilized inthe methods and compositions of the present invention. Examples ofadditional effector molecules are provided infra.

A polynucleotide sequence “corresponds” to a polypeptide sequence iftranslation of the polynucleotide sequence in accordance with thegenetic code yields the polypeptide sequence (i.e., the polynucleotidesequence “encodes” the polypeptide sequence), one polynucleotidesequence “corresponds” to another polynucleotide sequence if the twosequences encode the same polypeptide sequence.

A “heterologous” region or domain of a DNA construct is an identifiablesegment of DNA within a larger DNA molecule that is not found inassociation with the larger molecule in nature. Thus, when theheterologous region encodes a mammalian gene, the DNA flanking the geneusually does not flank the mammalian genomic DNA in the genome of thesource organism. Another example of a heterologous region is a constructwhere the coding sequence itself is not found in nature (e.g., a cDNAwhere the genomic coding sequence contains introns or syntheticsequences having codons different than the native gene). Allelicvariations or naturally-occurring mutational events do not give rise toa heterologous region of DNA as defined herein.

A “coding sequence” is an in-frame sequence of codons that correspond toor encode a protein or peptide sequence. Two coding sequences correspondto each other if the sequences or their complementary sequences encodethe same amino acid sequences. A coding sequence in association withappropriate regulatory sequences may be transcribed and translated intoa polypeptide. A polyadenylation signal and transcription terminationsequence will usually be located 3′ to the coding sequence. A “promotersequence” is a DNA regulatory region capable of initiating transcriptionof a downstream (3′ direction) coding sequence, and typically containadditional sites for binding of regulatory molecules, e.g.,transcription factors, that affect the transcription of the codingsequence. A coding sequence is “under the control” of the promotersequence or “operatively linked” to the promoter when RNA polymerasebinds the promoter sequence in a cell and transcribes the codingsequence into mRNA, which is then in turn translated into the proteinencoded by the coding sequence.

The general structure of antibodies in vertebrates now is wellunderstood (See Edelman, G. M., Ann. N.Y. Acad. Sci., 190:5 1971).Antibodies consist of two identical light polypeptide chains ofmolecular weight approximately 23,000 daltons (the “light chain”), andtwo identical heavy chains of molecular weight 53,000-70,000 (the “heavychain”). The four chains are joined by disulfide bonds in a “Y”configuration wherein the light chains bracket the heavy chains startingat the mouth of the “Y” configuration. The “branch” portion of the “Y”configuration is designated the F_(ab) region; the stem portion of the“Y” configuration is designated the F_(c) region. The amino acidsequence orientation runs from the N-terminal end at the top of the “Y”configuration to the C-terminal end at the bottom of each chain. TheN-terminal end possesses the variable region having specificity for theantigen that elicited it, and is approximately 100 amino acids inlength, there being slight variations between light and heavy chain andfrom antibody to antibody.

The variable region is linked in each chain to a constant region thatextends the remaining length of the chain and that within a particularclass of antibody does not vary with the specificity of the antibody(i.e., the antigen eliciting it). There are five known major classes ofconstant regions that determine the class of the immunoglobulin molecule(IgG, IgM, IgA, IgD, and IgE corresponding to γ, μ, α, δ, and ε (gamma,mu, alpha, delta, or epsilon) heavy chain constant regions). Theconstant region or class determines subsequent effector function of theantibody, including activation of complement (see Kabat, E. A.,Structural Concepts in Immunology and Immunochemistry, 2nd Ed., p.413-436, New York, N.Y.: Holt, Rinehart, Winston (1976)), and othercellular responses (see Andrews et al., Clinical Immunology, pp. 1-18,W. B. Sanders, Philadelphia, Pa. (1980); Kohl et al., Immunology, 48:187(1983)); while the variable region determines the antigen with which itwill react. Light chains are classified as either κ (kappa) or λ(lambda). Each heavy chain class can be prepared with either kappa orlambda light chain. The light and heavy chains are covalently bonded toeach other, and the “tail” portions of the two heavy chains are bondedto each other by covalent disulfide linkages when the immunoglobulinsare generated either by hybridomas or by B-cells.

The expression “variable region” or “VR” refers to the domains withineach pair of light and heavy chains in an antibody that are involveddirectly in binding the antibody to the antigen. Each heavy chain has atone end a variable domain (V_(H)) followed by a number of constantdomains. Each light chain has a variable domain (V_(L)) at one end and aconstant domain at its other end; the constant domain of the light chainis aligned with the first constant domain of the heavy chain, and thelight chain variable domain is aligned with the variable domain of theheavy chain.

The expressions “complementarity determining region,” “hypervariableregion,” or “CDR” refer to one or more of the hyper-variable orcomplementarity determining regions (“CDRs”) found in the variableregions of light or heavy chains of an antibody (See Kabat et al.,Sequences of Proteins of Immunological Interest, 4^(th) ed., Bethesda,Md.: U.S. Dept. of Health and Human Services, Public Health Service,National Institutes of Health (1987)). These expressions include thehypervariable regions as defined by Kabat et al., (Sequences of Proteinsof Immunological Interest, NIH Publication No. 91-3242, Bethesda, Md.:U.S. Dept. of Health and Human Services, National Institutes of Health(1983)) or the hypervariable loops in 3-dimensional structures ofantibodies (Chothia and Lesk, J. Mol. Biol., 196:901-917 1987). The CDRsin each chain are held in close proximity by framework regions (“FRs”)and, with the CDRs from the other chain, contribute to the formation ofthe antigen binding site. Within the CDRs there are select amino acidsthat have been described as the selectivity determining regions (“SDRs”)that represent the critical contact residues used by the CDR in theantibody-antigen interaction (see, Kashmiri et al., Methods,36(1):25-34, 2005).

An “epitope” or “binding site” is an area or region on an antigen towhich an antigen-binding peptide (such as an antibody) specificallybinds. A protein epitope may comprise amino acid residues directlyinvolved in the binding (also called immunodominant component of theepitope) and other amino acid residues, which are not directly involvedin the binding, such as amino acid residues that are effectively blockedby the specifically antigen binding peptide (in other words, the aminoacid residue is within the “footprint” of the specifically antigenbinding peptide). The term epitope herein includes both types of aminoacid binding sites in any particular region of PACAP, i.e., PACAP38 andPACAP27, that specifically binds to an anti-PACAP antibody. PACAP maycomprise a number of different epitopes, which may include, withoutlimitation, (1) linear peptide antigenic determinants, (2)conformational antigenic determinants that consist of one or morenon-contiguous amino acids located near each other in a mature PACAPconformation; and (3) post-translational antigenic determinants thatconsist, either in whole or part, of molecular structures covalentlyattached to a PACAP protein such as carbohydrate groups. In particular,the term “epitope” includes the specific residues in a protein orpeptide, e.g., PACAP, which are involved in the binding of an antibodyto such protein or peptide as determined by known and accepted methodssuch as alanine scanning techniques. Such methods are exemplifiedherein.

The phrase that an antibody (e.g., first antibody) binds “substantially”or “at least partially” the same epitope as another antibody (e.g.,second antibody) means that the epitope binding site for the firstantibody comprises at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,or more of the amino acid residues on the antigen that constitutes theepitope binding site of the second antibody. Also, that a first antibodybinds substantially or partially the same or overlapping epitope as asecond antibody means that the first and second antibodies compete inbinding to the antigen, as described above. Thus, the term “binds tosubstantially the same epitope or determinant as” a monoclonal antibodymeans that an antibody “competes” with the antibody.

The phrase “binds to the same or overlapping epitope or determinant as”an antibody of interest means that an antibody “competes” with saidantibody of interest for at least one, (e.g., at least 2, at least 3, atleast 4, at least 5) or all residues on PACAP to which said antibody ofinterest specifically binds. The identification of one or moreantibodies that bind(s) to substantially or essentially the same epitopeas the monoclonal antibodies described herein can be readily determinedusing alanine scanning. Additionally, any one of variety ofimmunological screening assays in which antibody competition can beassessed. A number of such assays are routinely practiced and well knownin the art (see, e.g., U.S. Pat. No. 5,660,827, issued Aug. 26, 1997,which is specifically incorporated herein by reference). It will beunderstood that actually determining the epitope to which an antibodydescribed herein binds is not in any way required to identify anantibody that binds to the same or substantially the same or overlappingepitope as the monoclonal antibody described herein.

For example, where the test antibodies to be examined are obtained fromdifferent source animals, or are even of a different Ig isotype, asimple competition assay may be employed in which the control antibodyis mixed with the test antibody and then applied to a sample containingPACAP. Protocols based upon ELISAs, radioimmunoassays, Western blotting,and the use of BIACORE® (GE Healthcare Life Sciences, Marlborough,Mass.) analysis are suitable for use in such simple competition studies.

In certain embodiments, the control anti-PACAP antibody is pre-mixedwith varying amounts of the test antibody (e.g., in ratios of about 1:1,1:2, 1:10, or about 1:100) for a period of time prior to applying to thePACAP38 or PACAP27 antigen sample. In other embodiments, the control andvarying amounts of test antibody can simply be added separately andadmixed during exposure to the PACAP38 or PACAP27 antigen sample. Aslong as bound antibodies can be distinguished from free antibodies(e.g., by using separation or washing techniques to eliminate unboundantibodies) and control antibody from the test antibody (e.g., by usingspecies specific or isotype specific secondary antibodies or byspecifically labeling the control antibody with a detectable label) itcan be determined if the test antibody reduces the binding of thecontrol antibody to the PACAP38 or PACAP27 antigens, indicating that thetest antibody recognizes substantially the same epitope as the controlanti-PACAP antibody. The binding of the (labeled) control antibody inthe presence of a completely irrelevant antibody (that does not bindPACAP) can serve as the control high value. The control low value can beobtained by incubating the labeled control antibody with the same butunlabeled control antibody, where competition would occur and reducebinding of the labeled antibody. In a test assay, a significantreduction in labeled antibody reactivity in the presence of a testantibody is indicative of a test antibody that recognizes substantiallythe same epitope, i.e., one that competes with the labeled controlantibody. For example, any test antibody that reduces the binding of thecontrol antibody to PACAP38 or PACAP27 by at least about 50%, such as atleast about 60%, or more preferably at least about 70% (e.g., about65-100%), at any ratio of test antibody between about 1:1 or 1:10 andabout 1:100 is considered to be an antibody that binds to substantiallythe same or overlapping epitope or determinant as the control antibody.

Preferably, such test antibody will reduce the binding of the controlantibody to PACAP38 or PACAP27 antigen preferably at least about 50%, atleast about 60%, at least about 80%, or at least about 90% (e.g., about95%) of the binding of the control antibody observed in the absence ofthe test antibody.

A simple competition assay in which a test antibody is applied atsaturating concentration to a surface onto which PACAP38 or PACAP27 isimmobilized also may be advantageously employed. The surface in thesimple competition assay is preferably a BIACORE® (GE Healthcare LifeSciences, Marlborough, Mass.) chip (or other media suitable for surfaceplasmon resonance (“SPR”) analysis). The binding of a control antibodythat binds PACAP38 or PACAP27 to the PACAP-coated surface is measured.This binding to the PACAP38- or PACAP27-containing surface of thecontrol antibody alone is compared with the binding of the controlantibody in the presence of a test antibody. A significant reduction inbinding to the PACAP38- or PACAP27-containing surface by the controlantibody in the presence of a test antibody indicates that the testantibody recognizes substantially the same epitope as the controlantibody such that the test antibody “competes” with the controlantibody. Any test antibody that reduces the binding of control antibodyby at least about 20% or more, at least about 40%, at least about 50%,at least about 70%, or more, can be considered to be an antibody thatbinds to substantially the same epitope or determinant as the controlantibody. Preferably, such test antibody will reduce the binding of thecontrol antibody to PACAP38 or PACAP27 by at least about 50% (e.g., atleast about 60%, at least about 70%, or more). It will be appreciatedthat the order of control and test antibodies can be reversed; i.e. thecontrol antibody can be first bound to the surface and then the testantibody is brought into contact with the surface thereafter in acompetition assay. Preferably, the “sandwich-style” binding assayexemplified in Example 9 infra is used. Alternatively, the antibodyhaving greater affinity for PACAP38 or PACAP27 antigen is bound to thePACAP38- or PACAP27-containing surface first, as it will be expectedthat the decrease in binding seen for the second antibody (assuming theantibodies are competing) will be of greater magnitude. Further examplesof such assays are provided in e.g., Saunal and Regenmortel, J. Immunol.Methods, 183:33-41 (1995), the disclosure of which is incorporatedherein by reference.

In addition, whether an antibody binds the same or overlappingepitope(s) on PACAP as another antibody or the epitope bound by a testantibody may in particular be determined using a Western-blot basedassay. In this assay a library of peptides corresponding to the antigenbound by the antibody, the PACAP protein, is made, that compriseoverlapping portions of the protein, typically 10-25, 10-20, or 10-15amino acids long. These different overlapping amino acid peptidesencompassing the PACAP sequence are synthesized and covalently bound toa PEPSPOTS' nitrocellulose membrane (JPT Peptide Technologies, Berlin,Germany). Blots are then prepared and probed according to themanufacturer's recommendations.

Essentially, the immunoblot assay then detects by fluorometric meanswhat peptides in the library bind to the test antibody and thereby canidentify what residues on the antigen, i.e., PACAP, interact with thetest antibody. (See U.S. Pat. No. 7,935,340, incorporated by referenceherein).

Various epitope mapping techniques are known in the art. By way ofexample, X-ray co-crystallography of the antigen and antibody; NMR; SPR(e.g., at 25° or 37° C.); array-based oligo-peptide scanning (or“pepscan analysis”); site-directed mutagenesis (e.g., alanine scanning);mutagenesis mapping; hydrogen-deuterium exchange; phage display; andlimited proteolysis are all epitope mapping techniques that are wellknown in the art (See, e.g., Epitope Mapping Protocols: Second Edition,Methods in Molecular Biology, editors Mike Schutkowski and UlrichReineke, 2^(nd) Ed., New York, N.Y.: Humana Press (2009), and EpitopeMapping Protocols, Methods in Molecular Biology, editor Glenn Morris,1^(st) Ed., New York, N.Y.: Humana Press (1996), both of which areherein incorporated by referenced in their entirety).

The identification of one or more antibodies that bind(s) tosubstantially or essentially the same epitope as the monoclonalantibodies described herein, e.g., Ab10 or Ab20, can be readilydetermined using any one of variety of immunological screening assays inwhich antibody competition can be assessed. A number of such assays areroutinely practiced and well known in the art (see, e.g., U.S. Pat. No.5,660,827, incorporated herein by reference). It will be understood thatdetermining the epitope to which an antibody described herein binds isnot in any way required to identify an antibody that binds to the sameor substantially the same epitope as the monoclonal antibody describedherein.

For example, where the test antibodies to be examined are obtained fromdifferent source animals, or are even of a different Ig isotype, asimple competition assay may be employed in which the control antibody(one of Ab10 or Ab20, for example) is mixed with the test antibody andthen applied to a sample containing either or both PACAP38 and PACAP27,each of which is known to be bound by Ab10 or Ab20. Protocols based uponELISAs, radioimmunoassays, Western blotting, and BIACORE® (GE HealthcareLife Sciences, Marlborough, Mass.) analysis (as described in theExamples section herein) are suitable for use in such simple competitionstudies.

In certain embodiments, the method comprises pre-mixing the controlantibody with varying amounts of the test antibody (e.g., in ratios ofabout 1:1, 1:2, 1:10, or about 1:100) for a period of time prior toapplying to the PACAP antigen sample. In other embodiments, the controland varying amounts of test antibody can be added separately and admixedduring exposure to the PACAP antigen sample. As long as bound antibodiescan be distinguished from free antibodies (e.g., by using separation orwashing techniques to eliminate unbound antibodies) and control antibodyfrom the test antibody (e.g., by using species specific or isotypespecific secondary antibodies or by specifically labelling the controlantibody with a detectable label), the method can be used to determinethat the test antibody reduces the binding of the control antibody tothe PACAP antigen, indicating that the test antibody recognizessubstantially the same epitope as the control antibody (e.g., Ab10 orAb20). The binding of the (labeled) control antibody in the presence ofa completely irrelevant antibody (that does not bind PACAP) can serve asthe control high value. The control low value can be obtained byincubating the labeled control antibody with the same but unlabeledcontrol antibody, where competition would occur and reduce binding ofthe labeled antibody. In a test assay, a significant reduction inlabeled antibody reactivity in the presence of a test antibody isindicative of a test antibody that recognizes substantially the sameepitope, i.e., one that competes with the labeled control antibody. Forexample, any test antibody that reduces the binding of Ab10 or Ab20 toboth of PACAP38 and PACAP27 antigens by at least about 50%, such as atleast about 60%, or more preferably at least about 70% (e.g., about65-100%), at any ratio of control Ab10 or Ab20:test antibody, or Ab10 orAb20:test antibody between about 1:1 or 1:10 and about 1:100 isconsidered to be an antibody that binds to substantially the sameepitope or determinant as Ab10 or Ab20, respectively. Preferably, suchtest antibody will reduce the binding of Ab10 or Ab20 to at least one,preferably each, of the PACAP38 and PACAP27 antigens preferably at leastabout 50%, at least about 60%, at least about 80% or at least about 90%(e.g., about 95%) of the binding of Ab10 or Ab20 observed in the absenceof the test antibody. These methods can be adapted to identify and/orevaluate antibodies that compete with other control antibodies.

A simple competition assay in which a test antibody is applied atsaturating concentration to a surface onto which either PACAP38 orPACAP27, or both, are immobilized also may be advantageously employed.The surface in the simple competition assay is preferably of a mediasuitable for OCTET® and/or PROTEON®. The binding of a control antibody(e.g., Ab10 or Ab20) to the PACAP-coated surface is measured. Thisbinding to the PACAP-containing surface of the control antibody alone iscompared with the binding of the control antibody in the presence of atest antibody. A significant reduction in binding to thePACAP-containing surface by the control antibody in the presence of atest antibody indicates that the test antibody recognizes substantiallythe same epitope as the control antibody such that the test antibody“competes” with the control antibody. Any test antibody that reduces thebinding of control antibody (such as Ab10 or Ab20) to both of PACAP38and PACAP27 antigens by at least about 20% or more, at least about 40%,at least about 50%, at least about 70%, or more, can be considered to bean antibody that binds to substantially the same epitope or determinantas the control antibody (e.g., Ab10 or Ab20). Preferably, such testantibody will reduce the binding of the control antibody (e.g., Ab10 orAb20) to the PACAP antigen by at least about 50% (e.g., at least about60%, at least about 70%, or more). It will be appreciated that the orderof control and test antibodies can be reversed; i.e. the controlantibody can be first bound to the surface and then the test antibody isbrought into contact with the surface thereafter in a competition assay.Preferably, the antibody having higher affinity for PACAP38 and PACAP27is bound to the PACAP-containing surface first, as it will be expectedthat the decrease in binding seen for the second antibody (assuming theantibodies are competing) will be of greater magnitude. Further examplesof such assays are provided in, e.g., Saunal and Regenmortel, J.Immunol. Methods, 183:33-41 (1989), the disclosure of which isincorporated herein by reference.

Determination of whether an antibody, antigen binding fragment thereof,or antibody derivative binds within one of the epitope regions definedabove can be carried out in ways known to the person skilled in the art.In another example of such mapping/characterization methods, an epitoperegion for an anti-PACAP antibody may be determined by epitope“footprinting” using chemical modification of the exposedamines/carboxyls in the PACAP38 and PACAP27 protein. One specificexample of such a foot-printing technique is the use ofhydrogen-deuterium exchange detected by mass spectrometry (“HXMS”),wherein a hydrogen/deuterium exchange of receptor and ligand proteinamide protons, binding, and back exchange occurs, wherein the backboneamide groups participating in protein binding are protected from backexchange and therefore will remain deuterated. Relevant regions can beidentified at this point by peptic proteolysis, fast microborehigh-performance liquid chromatography separation, and/or electrosprayionization mass spectrometry (See, e.g., Ehring H., Anal. Biochem.,267(2):252-259, 1999; and Engen, J. R. and Smith, D. L., Anal. Chem.,73:256A-265A, 2001). Another example of a suitable epitopeidentification technique is nuclear magnetic resonance epitope mapping(“NMR”), where typically the position of the signals in two-dimensionalNMR spectres of the free antigen and the antigen complexed with theantigen binding peptide, such as an antibody, are compared. The antigentypically is selectively isotopically labeled with ¹⁵N so that onlysignals corresponding to the antigen and no signals from the antigenbinding peptide are seen in the NMR-spectrum. Antigen signalsoriginating from amino acids involved in the interaction with theantigen binding peptide typically will shift position in the spectres ofthe complex compared to the spectres of the free antigen, and the aminoacids involved in the binding can be identified that way. See, e.g.,Ernst Schering Res. Found. Workshop, (44):149-67, 2004; Huang et al., J.Mol. Biol., 281(1):61-67, 1998; and Saito and Patterson, Methods,9(3):516-24, 1996).

Epitope mapping/characterization also can be performed using massspectrometry (“MS”) methods (See, e.g., Downard, J. Mass Spectrom.,35(4):493-503, 2000; and Kiselar and Downard, Anal. Chem.,71(9):1792-801, 1999).

Protease digestion techniques also can be useful in the context ofepitope mapping and identification. Antigenic determinant-relevantregions/sequences can be determined by protease digestion, e.g. by usingtrypsin in a ratio of about 1:50 to PACAP38 or PACAP27 overnight (“o/n”)digestion at 37° C. and pH 7-8, followed by mass spectrometry (“MS”)analysis for peptide identification. The peptides protected from trypsincleavage by the anti-PACAP antibody can subsequently be identified bycomparison of samples subjected to trypsin digestion and samplesincubated with antibody and then subjected to digestion by e.g. trypsin(thereby revealing a footprint for the antibody). Other enzymes likechymotrypsin or pepsin can be used in similar epitope characterizationmethods. Moreover, enzymatic digestion can provide a quick method foranalyzing whether a potential antigenic determinant sequence is within aregion of PACAP in the context of a PACAP-binding polypeptide. If thepolypeptide is not surface exposed, it is most likely not relevant interms of immunogenicity/antigenicity (See, e.g., Manca, Ann. 1st. Super.Sanità, 27(1):15-9, 1991, for a discussion of similar techniques).

Site-directed mutagenesis is another technique useful forcharacterization of a binding epitope. For example, in“alanine-scanning” site-directed mutagenesis (also known as alaninescanning, alanine scanning mutagenesis, alanine scanning mutations,combinatorial alanine scanning, or creation of alanine point mutations,for example), each residue within a protein segment is replaced with analanine residue (or another residue such as valine where alanine ispresent in the wild-type sequence) through such methodologies as directpeptide or protein synthesis, site-directed mutagenesis, the GENEART™Mutagenesis Service (Thermo Fisher Scientific, Waltham, Mass. U.S.A.) orshotgun mutagenesis, for example. A series of single point mutants ofthe molecule is thereby generated using this technique; the number ofmutants generated is equivalent to the number of residues in themolecule, each residue being replaced, one at a time, by a singlealanine residue. Alanine is generally used to replace native (wild-type)residues because of its non-bulky, chemically inert, methyl functionalgroup that can mimic the secondary structure preferences that many otheramino acids may possess. Subsequently, the effects replacing a nativeresidue with an alanine has on binding affinity of an alanine scanningmutant and its binding partner can be measured using such methods as,but not limited to, SPR binding experiments. If a mutation leads to asignificant reduction in binding affinity, it is most likely that themutated residue is involved in binding. Monoclonal antibodies specificfor structural epitopes (i.e., antibodies that do not bind the unfoldedprotein) can be used as a positive control for binding affinityexperiments to verify that the alanine-replacement does not influencethe overall tertiary structure of the protein (as changes to the overallfold of the protein may indirectly affect binding and thereby produce afalse positive result) (See, e.g., Clackson and Wells, Science,267:383-386, 1995; Weiss et al., Proc. Natl. Acad. Sci. USA,97(16):8950-8954, 2000; and Wells, Proc. Natl. Acad. Sci. USA, 93:1-6,1996). In Example 12 alanine scanning methods are used to identify thespecific epitope or residues of PACAP which specifically interact withthe anti-PACAP antibodies disclosed herein.

Electron microscopy can also be used for epitope “footprinting”. Forexample, Wang et al., Nature, 355:275-278 (1992) used coordinatedapplication of cryoelectron microscopy, three-dimensional imagereconstruction, and X-ray crystallography to determine the physicalfootprint of a Fab-fragment on the capsid surface of native cowpeamosaic virus.

Other forms of “label-free” assay for epitope evaluation include SPR(sold commercially as the BIACORE® system, GE Healthcare Life Sciences,Marlborough, Mass.) and reflectometric interference spectroscopy(“RifS”) (See, e.g., Fagerstam et al., J. Mol. Recog., 3:208-14, 1990;Nice et al., J. Chromatogr., 646:159-168, 1993; Leipert et al., Angew.Chem. Int. Ed., 37:3308-3311, 1998; Kroger et al., Biosensors andBioelectronics, 17:937-944, 2002).

The expressions “framework region” or “FR” refer to one or more of theframework regions within the variable regions of the light and heavychains of an antibody (See Kabat et al., Sequences of Proteins ofImmunological Interest, 4^(th) edition, Bethesda, Md.: U.S. Dept. ofHealth and Human Services, Public Health Service, National Institutes ofHealth, 1987). These expressions include those amino acid sequenceregions interposed between the CDRs within the variable regions of thelight and heavy chains of an antibody.

The term “Fc region” is used to define a C-terminal region of animmunoglobulin heavy chain. The “Fc region” may be a native sequence Fcregion or a variant Fc region. Although the boundaries of the Fc regionof an immunoglobulin heavy chain might vary, the human IgG heavy chainFc region is usually defined to stretch from an amino acid residue atposition Cys226, or from Pro230, to the carboxyl-terminus thereof. Thenumbering of the residues in the Fc region is that of the EU index as inKabat (See, Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th edition, Bethesda, Md.: U.S. Dept. of Health and HumanServices, Public Health Service, National Institutes of Health, 1991).The Fc region of an immunoglobulin generally comprises two constantdomains, CH2 and CH3.

The terms “Fc receptor” and “FcR” describe a receptor that binds to theFc region of an antibody. The preferred FcR is a native sequence humanFcR. Moreover, a preferred FcR is one that binds an IgG antibody (agamma receptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof these receptors. FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. FcRs are reviewed in Ravetch and Kinet, Ann. Rev. Immunol.,9:457-92 (1991); Capel et al., Immunomethods, 4:25-34 (1994); and deHaas et al., J. Lab. Clin. Med., 126:330-41 (1995). “FcR” also includesthe neonatal receptor, FcRn, which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol., 117:587, 1976;and Kim et al., J. Immunol., 24:249, 1994), and which primarilyfunctions to modulate and/or extend the half-life of antibodies incirculation. To the extent that the disclosed anti-PACAP antibodies areaglycosylated, as a result of the expression system and/or sequence, thesubject antibodies are expected to bind FcRn receptors, but not to bind(or to minimally bind) Fcγ receptors.

A “functional Fc region” possesses at least one effector function of anative sequence Fc region. Exemplary “effector functions” include C1qbinding; complement dependent cytotoxicity (“CDC”); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (“ADCC”); phagocytosis;down-regulation of cell surface receptors (e.g. B cell receptor(“BCR”)), etc. Such effector functions generally require the Fc regionto be combined with a binding domain (e.g. an antibody variable domain)and can be assessed using various assays known in the art for evaluatingsuch antibody effector functions.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. A “variantFc region” comprises an amino acid sequence that differs from that of anative sequence Fc region by virtue of at least one amino acidmodification, yet retains at least one effector function of the nativesequence Fc region. Preferably, the variant Fc region has at least oneamino acid substitution compared to a native sequence Fc region or tothe Fc region of a parent polypeptide, e.g. from about one to about tenamino acid substitutions, and preferably from about one to about fiveamino acid substitutions in a native sequence Fc region or in the Fcregion of the parent polypeptide. The variant Fc region herein willpreferably possess at least about 80% sequence identity with a nativesequence Fc region and/or with an Fc region of a parent polypeptide, andmost preferably at least about 90% sequence identity therewith, morepreferably at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99% sequence identity therewith.

Anti-PA CAP Antibodies and Binding Fragments Thereof Having BindingActivity for PACAP

PACAP is a multifunctional vasodilatory peptide with expressionthroughout the central nervous system (“CNS”) and periphery. PACAP is amember of the secretin/VIP/GRH family. PACAP exists in two α-amidatedactive forms, PACAP38 (SEQ ID NO: 1241) and PACAP27 (SEQ ID NO: 1242).Herein, the term “PACAP” includes either or both of PACAP38 and PACAP27unless expressly indicated otherwise. PACAP is highly conserved betweenspecies.

In humans, PACAP is derived from a 176 amino acid precursor protein(preproPACAP) and the gene is located on chromosome 18p11, with PACAP38encoded for by exon 5 (See, Vaudry et al., Pharmacol. Rev., 61:283-357,2009). PreproPACAP contains an N-terminal 24 amino acid signal protein,a 29 amino acid PACAP-related peptide and PACAP in the C-terminaldomain. The precursor is metabolized by prohormone convertase enzymesinto biologically active PACAP38 and PACAP27.

VIP (SEQ ID NO: 1243) belongs to the same protein family as PACAP andshares high homology with PACAP, i.e., VIP and PACAP27 have 68% sequencehomology at the amino acid level, as well as similar overall secondarystructure, i.e. long alpha-helical structures at the C-terminus.

PACAP's actions are mediated via three different G-protein coupledreceptors: PAC1-R, VPAC1-R, and VPAC2-R. VPAC1-R can associate with allof the receptor-associated membrane proteins (“RAMPs”, see Kaiser andRusso, Neuropeptides, 47:451-461, 2013). PAC1-R is selective for PACAP,whereas VPAC1-R and VPAC2-R bind to both VIP and PACAP with highaffinity. PAC1-R binds to PACAP with 100-1000-fold higher affinity thanVIP, i.e., K_(D)˜0.5 nM for PACAP27/PACAP38 vs. K_(D)˜500 nM for VIP.Conversely, VPAC1-R and VPAC2-R have equal affinities for PACAP and VIP(K_(D)˜1 nM), (See, Schytz et al. 2010). All three receptors are widelyexpressed in both peripheral tissues and in the CNS, with PAC1-Rpredominantly expressed in the CNS, most abundantly in the olfactorybulb, thalamus, hypothalamus, the dentate gyrus of the hippocampus andin granule cells of the cerebellum (See, Hashimoto et al., J. Comp.Neurol., 371:567-577, 1996; Shioda et al., Neurosci. Res., 28:345-354,1997).

Activation of the PAC1-R, VPAC1-R, and/or VPAC2-R results in increasedadenylate cyclase activity and, thus, increased cAMP production.However, PACAP receptors can also mediate their effects through PLC,leading to increased Ca²⁺ levels, and PLD.

PACAP has a wide range of biological effects, including a role inneurodevelopment, neuroprotection, neuromodulation, neurogenicinflammation, and nociception. PACAP is also reported to interact withglycosaminoglycans (“GAGs”). GAGs are long, unbranched polysaccharidescomposed of repeating disaccharide units, such as heparin, chondroitin,keratin, and hyaluronic acid. It has been shown that the cellular uptakeof PACAP is dependent on the expression of GAG proteins and that PACAPbound to sulfated GAGs. Particularly, it was determined that PACAP38binding to GAGs was capable of inducing receptor-independent cellularuptake of PACAP38. This study further demonstrated that a randomcoil-to-α-helix transition in PACAP38 was essential for GAG-dependentuptake of PACAP38, as a mutant PACAP38 that could not undergo thestructural transition was not internalized by GAG-containing cell linesas efficiently as the wild-type form of PACAP38 (Neree et al., FEBSLett., 588(24):4590-4596, 2014). In a follow up study, it was determinedthat PACAP's ability to cluster GAGs, i.e., heparin, was directlyrelated to its ability to function as a cell penetrating peptide(“CPP”). It is hypothesized that this activity is attributable to theheparin-binding, or Cardin-Weintraub, motif found insecretin/glucagon/GHRH family members, such as PACAP (Neree et al., Int.J. Mol. Sci., 16:27391-27400, 2015). Interestingly, Neree et al. (2015)presented data demonstrating that PACAP38 was able to cluster sulfatedGAGs in vitro. These data suggested that the observed clustering effectis important for the GAG-mediated cellular uptake of PACAP38, as otherpeptides, such as glucagon, displayed higher binding affinities forsulfated GAGs (heparin) but are not internalized by cells as efficientlyas PACAP38. Further, it is reported that in in vitro studies in whichcells are exposed to PACAP, cartilage formation is increased, includingcartilage matrix that is rich in sulphated GAG proteins, consistent withits putative protective role expressed during various cellular stressresponses (Juhász et al., PLoS ONE, 9(3):e91541, 2014). Using cell typesthat lack PACAP-specific receptors on their plasma membranes, such asCHO-K1 cells, Doan et al. presented data demonstrating the ability ofsuch cells to engage in receptor-independent cellular uptake of variousforms of fluorescently-labeled PACAP38 and PACAP27 (Doan et al.,Biochem. Biophys. Acta, 1823:940-949, 2012).

The present invention provides exemplary antibodies or antigen bindingfragments thereof that bind PACAP, including human PACAP. Otherantibodies or antigen binding fragments thereof that bind PACAP,including those having different CDRs, and epitopic specificity may beobtained using the disclosure of the present specification, and usingmethods that are generally known in the art. Such antibodies and antigenbinding fragments thereof antagonize the biological effects of PACAP invivo and therefore are useful in treating or preventing PACAP-relatedconditions including, for example, headache, migraine, pain,photophobia, hot flush, PTSD, and anxiety disorders. In preferredembodiments, the antibody or antigen binding fragment thereof accordingto the invention comprises one or more CDRs, a V_(L) chain and/or V_(H)chain of the anti-PACAP antibodies and antigen binding fragments thereofdescribed herein.

In some embodiments, an anti-PACAP antibody or antigen binding fragmentthereof according to the invention will interfere with, block, reduce,or modulate the interaction between PACAP and its receptor(s) (e.g.,PAC1-R, VPAC1-R, and VPAC2-R). In some instances an anti-PACAP antibodyor antigen binding fragment thereof according to the invention is“neutralizing”, e.g., it totally prevents the specific interaction ofPACAP with PAC1-R, VPAC1-R, and/or VPAC2-R. In some embodiments, theantibody or antigen binding fragment thereof neutralizes PACAP, e.g., byremaining bound to PACAP in a location and/or manner that prevents PACAPfrom specifically binding to PAC1-R, VPAC1-R, and/or VPAC2-R.

In some embodiments, the antibody or antigen binding fragment thereofaccording to the invention is capable of inhibiting PACAP-mediatedactivity (including binding to PAC1-R-expressing cells). In someembodiments, the antibody or antigen binding fragment thereof accordingto the invention are humanized, such as humanized rabbit antibodies toPACAP.

As mentioned, the anti-PACAP antibodies or antigen binding fragmentsthereof according to the invention have a variety of uses. For example,the subject antibodies and fragments can be useful in therapeuticapplications, as well as diagnostically in binding assays. The subjectanti-PACAP antibodies or antigen binding fragments thereof are usefulfor affinity purification of PACAP, in particular human PACAP or itsligands and in screening assays to identify other antagonists of PACAPactivity. Some of the antibodies or antigen binding fragments thereofare useful for inhibiting binding of PACAP to PAC1-R, VPAC1-R, and/orVPAC2-R, or inhibiting PACAP-mediated activities and/or biologicaleffects.

As used herein, the term “one or more biological effects associated withPACAP refers to any biological effect mediated, induced, or otherwiseattributable to PACAP, e.g., binding properties, functional properties,and other properties of biological significance. Non-limiting exemplarybiological effects of PACAP include PACAP binding to PAC1-R, VPAC1-R,and/or VPAC2-R; PACAP activating PAC1-R, VPAC1-R, and/orVPAC2-R-mediated signaling; PACAP-mediated increase in cAMP production;PACAP-mediated increase in PLC activity; PACAP-mediated increase in PLDactivity; PACAP-mediated increase in Ca²⁺ levels; and PACAP-mediatedvasodilation, photophobia, mast cell degranulation, and/or neuronalactivation. The subject anti-PACAP antibodies are capable of inhibitingone, a combination of, or all of these exemplary PACAP biologicalactivities. For example, the anti-PACAP antibodies and antigen bindingfragments thereof provided herein are capable of inhibitingPACAP-induced vasodilation (see Example 7 and Example 8).

The antibody or antigen binding fragment thereof according to theinvention can be used in a variety of therapeutic applications. Forexample, in some embodiments the anti-PACAP antibody or antigen bindingfragment thereof are useful for treating conditions associated withPACAP, such as, but not limited to, migraine (with or without aura),hemiplegic migraines, cluster headaches, migrainous neuralgia, chronicheadaches, tension headaches, general headaches, hot flush, photophobia,chronic paroxysmal hemicrania, secondary headaches due to an underlyingstructural problem in the head or neck, cranial neuralgia, sinusheadaches (e.g., headache associated with sinusitis), allergy-inducedheadaches or migraines, pain, chronic pain, neuroinflammatory orinflammatory pain, post-operative incision pain, post-surgical pain,trauma-related pain, eye pain, tooth pain, complex regional painsyndrome, cancer pain (e.g., primary or metastatic bone cancer pain),fracture pain, osteoporotic fracture pain, pain resulting from burn,gout joint pain, pain associated with sickle cell crises, painassociated with temporomandibular disorders, cirrhosis, hepatitis,neurogenic pain, neuropathic pain, nociceptic pain, visceral pain,trigeminal neuralgia, post-herpetic neuralgia, phantom limb pain,fibromyalgia, menstrual pain, ovarialgia, reflex sympathetic dystrophy,osteoarthritis or rheumatoid arthritis pain, lower back pain, diabeticneuropathy, sciatica, dyspepsia, irritable bowel syndrome, inflammatorybowel disease, Crohn's disease, ileitis, ulcerative colitis, renalcolic, dysmenorrhea, cystitis, interstitial cystitis, menstrual period,labor, menopause, pancreatitis, schizophrenia, depression, PTSD, anxietydisorders, diabetes, autoimmune diabetes, endothelial dysfunction,ischemia, Raynaud's syndrome, coronary heart disease (“CHD”), coronaryartery disease (“CAD”), heart failure, peripheral arterial disease(“PAD”), pulmonary hypertension (“PH”), connective tissue disorders,stroke, Sjögren's syndrome, multiple sclerosis, bronchialhyperreactivity, asthma, bronchitis, bronchodilation, emphysema, chronicobstructive pulmonary disease (“COPD”), inflammatory dermatitis,adenocarcinoma in glandular tissue, blastoma in embryonic tissue oforgans, carcinoma in epithelial tissue, leukemia in tissues that formblood cells, lymphoma in lymphatic tissue, myeloma in bone marrow,sarcoma in connective or supportive tissue, adrenal cancer, AIDS-relatedlymphoma, anemia, bladder cancer, bone cancer, brain cancer, breastcancer, carcinoid tumors, cervical cancer, chemotherapy, colon cancer,cytopenia, endometrial cancer, esophageal cancer, gastric cancer, headcancer, neck cancer, hepatobiliary cancer, kidney cancer, leukemia,liver cancer, lung cancer, lymphoma, Hodgkin's disease, non-Hodgkin's,nervous system tumors, oral cancer, ovarian cancer, pancreatic cancer,prostate cancer, rectal cancer, skin cancer, stomach cancer, testicularcancer, thyroid cancer, urethral cancer, cancer of bone marrow, multiplemyeloma, tumors that metastasize to the bone, tumors infiltrating thenerve and hollow viscus, tumors near neural structures, acne vulgaris,atopic dermatitis, urticaria, keloids, hypertrophic scars and rosacea,allergic dermatitis, psoriasis, pruritus, neurogenic cutaneous redness,erythema, weight loss, anorexia, sarcoidosis, shock, sepsis, opiatewithdrawal syndrome, morphine tolerance, epilepsy, LUT disorders such asurinary tract infection, abnormal voiding, urinary urgency, nocturia,urinary incontinence, overactive bladder, and for preventing oralleviating the pain associated with such LUT conditions.

Specific examples of visceral pain, i.e., pain associated with theviscera, or the internal organs of the body include pain that affectsorgans such as e.g., the heart, lungs, reproductive organs, bladder,ureters, the digestive organs, liver, pancreas, spleen, and kidneys.Conditions associated therewith include by way of example pancreatitis,labor, abdominal surgery associated with ileus, cystitis, menstrualperiod, or dysmenorrhea. Likewise, kidney pain, epigastric pain, pleuralpain, and painful biliary colic, appendicitis pain may all be consideredto be visceral pain. Substernal pain or pressure from early myocardialinfarction is also visceral. Diseases of the stomach, duodenum or coloncan cause visceral pain. Commonly encountered gastrointestinal (“GI”)disorders that cause visceral pain include functional bowel disorder(“FBD”) and inflammatory bowel disease (“IBD”). Such GI disorders mayfurther include gastro-esophageal reflux, dyspepsia, irritable bowelsyndrome (“IBS”) and functional abdominal pain syndrome (“FAPS”), and,with respect to IBD, Crohn's disease, ileitis, and ulcerative colitis.

The subject anti-PACAP antibodies and antigen binding fragments thereofmay be used alone or in association with other active agents or drugs,including other biologics, to treat any subject in which blocking,inhibiting, or neutralizing the in vivo effect of PACAP or blocking orinhibiting the interaction of PACAP and its receptors, PAC1-R, VPAC1-R,and VPAC2-R, is therapeutically desirable.

Exemplary anti-PACAP antibodies and antigen binding fragments thereofaccording to the invention, and the specific CDRs thereof are identifiedin this section. For convenience, each exemplified antibody or antigenbinding fragment thereof, and corresponding sequences are separatelyidentified by a specific nomenclature, i.e., Ab10 or Ab20.

The anti-PACAP antibodies and antigen binding fragments thereofcomprising the invention have binding affinity for PACAP, wherein thebinding affinity comprises anti-PACAP antibodies or antigen bindingfragments thereof specifically binding to PACAP38 and PACAP27, but notbinding VIP, and/or antibodies or antigen binding fragments thereofspecifically binding to PACAP38, but not binding to PACAP27 or VIP,and/or antibodies or antigen binding fragments thereof specificallybinding to a linear and/or conformational epitope within PACAP38 and/orPACAP27. More specifically, the epitopes of PACAP38 and/or PACAP27 towhich antagonistic anti-PACAP antibodies or antigen binding fragmentsthereof according to the invention bind will include those which areidentified in Example 12 or residues thereof (as determined by use ofalanine scanning) and/or other epitopic identification methods.

Anti-PACAP Antibody Polypeptide Sequences

Antibody Ab10

In one embodiment, the invention includes antibodies and antigen-bindingfragments having binding specificity to PACAP that possess a heavy chainsequence comprising the sequence of SEQ ID NO: 401 which consists of theheavy chain variable region of SEQ ID NO: 402 linked to the heavy chainconstant region of SEQ ID NO: 410.

In one embodiment, the invention includes antibodies and antigen-bindingfragments having binding specificity to PACAP that contain a variableheavy chain sequence comprising the sequence set forth below:

(SEQ ID NO: 402) QSVEESGGRLVTPGTPLTLTCTVSGIDLNSYYMTWVRQAPGKGLEWIGFIDAGGDAYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARDLDLWG QGTLVTVSS.

In another embodiment, the invention includes antibodies andantigen-binding fragments having binding specificity to PACAP that bindthe same epitope as Ab10, and that contain a constant heavy chainsequence comprising the polypeptide of SEQ ID NO: 1244, 1245, or 1246,or comprising the sequence set forth below:

(SEQ ID NO: 410) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In another embodiment, the invention includes antibodies andantigen-binding fragments having binding specificity to PACAP thatcontain a light chain sequence comprising the sequence of SEQ ID NO: 421which consists of the light chain variable region of SEQ ID NO: 422linked to the light chain constant region of SEQ ID NO: 430.

In another embodiment, the invention includes antibodies andantigen-binding fragments having binding specificity to PACAP thatcontain a variable light chain sequence comprising the sequence setforth below:

(SEQ ID NO: 422) AAVLTQTPSPVSAAVGGTVTINCQSSESVYGNYLAWFQQKPGQPPKLLIYEASKLESGVPSRFSGSGSGTQFTLTISDLQCDDAATYYCAGGDISEGVAF GGGTEVVVKR.

In another embodiment, the invention includes antibodies andantigen-binding fragments having binding specificity to PACAP, that bindthe same epitope as Ab10, and that contain a constant light chainsequence comprising the sequence set forth below:

(SEQ ID NO: 430) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC.

In another embodiment, the invention includes antibodies andantigen-binding fragments having binding specificity to PACAP thatcontain one, two, or three of the polypeptide sequences of SEQ ID NO:404; SEQ ID NO: 406; and SEQ ID NO: 408, which correspond to the CDRs(hypervariable regions) of the heavy chain sequence of SEQ ID NO: 401,or which contain the variable heavy chain sequence of SEQ ID NO: 402,and/or which further contain one, two, or three of the polypeptidesequences of SEQ ID NO: 424; SEQ ID NO: 426; and SEQ ID NO: 428, whichcorrespond to the CDRs (hypervariable regions) of the light chainsequence of SEQ ID NO: 421, or which contain the variable light chainsequence of SEQ ID NO: 422, or antibodies or antigen-binding fragmentscontaining combinations of sequences that are at least 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99% identical thereto. In another embodiment ofthe invention, the antibodies of the invention and antigen-bindingfragments comprise, or alternatively consist of, combinations of one ormore of the exemplified variable heavy chain and variable light chainsequences, or the heavy chain and light chain sequences set forth above,or sequences that are at least 90% or 95% identical thereto.

The invention further contemplates anti-PACAP antibodies andantigen-binding fragments comprising one, two, three, or four of thepolypeptide sequences of SEQ ID NO: 403; SEQ ID NO: 405; SEQ ID NO: 407;and SEQ ID NO: 409, which correspond to the FRs (constant regions) ofthe heavy chain sequence of SEQ ID NO: 401, or the variable heavy chainsequence of SEQ ID NO: 402, and/or one, two, three, or four of thepolypeptide sequences of SEQ ID NO: 423; SEQ ID NO: 425; SEQ ID NO: 427;and SEQ ID NO: 429, which correspond to the FRs (constant regions) ofthe light chain sequence of SEQ ID NO: 421, or the variable light chainsequence of SEQ ID NO: 422, or combinations of these polypeptidesequences, or sequences that are at least 80%, 90%, 95%, 96%, 97%, 98%,or 99% identical therewith.

In another embodiment of the invention, the anti-PACAP antibodies andantigen-binding fragments of the invention or fragments comprise, oralternatively consist of, combinations of one or more of the FRs, CDRs,the variable heavy chain and variable light chain sequences, and theheavy chain and light chain sequences set forth above, including all ofthem, or sequences that are at least 90% or 95% identical thereto.

In another embodiment of the invention, the anti-PACAP antibodies andantigen-binding fragments of the invention comprise, or alternativelyconsist of, the polypeptide sequence of SEQ ID NO: 401, or SEQ ID NO:402, or polypeptides that are at least 90% or 95% identical thereto. Inanother embodiment of the invention, the antibodies and antigen-bindingfragments of the invention comprise, or alternatively consist of, thepolypeptide sequence of SEQ ID NO: 421, or SEQ ID NO: 422, orpolypeptides that are at least 90% or 95% identical thereto.

In a further embodiment of the invention, the antibodies andantigen-binding fragments having binding specificity to PACAP comprise,or alternatively consist of, one, two, or three of the polypeptidesequences of SEQ ID NO: 404; SEQ ID NO: 406; and SEQ ID NO: 408, whichcorrespond to the CDRs (hypervariable regions) of the heavy chainsequence of SEQ ID NO: 401, or the variable heavy chain sequence of SEQID NO: 402, or sequences that are at least 90% or 95% identical thereto.

In a further embodiment of the invention, the antibodies andantigen-binding fragments having binding specificity to PACAP comprise,or alternatively consist of, one, two, or three of the polypeptidesequences of SEQ ID NO: 424; SEQ ID NO: 426; and SEQ ID NO: 428, whichcorrespond to the CDRs (hypervariable regions) of the light chainsequence of SEQ ID NO: 421, or the variable light chain sequence of SEQID NO: 422, or sequences that are at least 90% or 95% identical thereto.

In a further embodiment of the invention, the antibodies andantigen-binding fragments having binding specificity to PACAP comprise,or alternatively consist of, one, two, three, or four of the polypeptidesequences of SEQ ID NO: 403; SEQ ID NO: 405; SEQ ID NO: 407; and SEQ IDNO: 409, which correspond to the FRs (constant regions) of the heavychain sequence of SEQ ID NO: 401, or the variable heavy chain sequenceof SEQ ID NO: 402, or sequences that are at least 90% or 95% identicalthereto.

In a further embodiment of the invention, the subject antibodies andantigen-binding fragments having binding specificity to PACAP comprise,or alternatively consist of, one, two, three, or four of the polypeptidesequences of SEQ ID NO: 423; SEQ ID NO: 425; SEQ ID NO: 427; and SEQ IDNO: 429, which correspond to the FRs (constant regions) of the lightchain sequence of SEQ ID NO: 421, or the variable light chain sequenceof SEQ ID NO: 422, or sequences that are at least 90% or 95% identicalthereto.

The invention also contemplates anti-PACAP antibodies andantigen-binding fragments that include one or more of the antibodyfragments described herein. In one embodiment of the invention,antibodies and antigen-binding fragments having binding specificity toPACAP comprise, or alternatively consist of, one, two, three, or more,including all of the following antibody fragments: the variable heavychain region of SEQ ID NO: 402; the variable light chain region of SEQID NO: 422; the complementarity determining regions (SEQ ID NO: 404; SEQID NO: 406; and SEQ ID NO: 408) of the variable heavy chain region ofSEQ ID NO: 402; and the complementarity determining regions (SEQ ID NO:424; SEQ ID NO: 426; and SEQ ID NO: 428) of the variable light chainregion of SEQ ID NO: 422, or sequences that are at least 90% or 95%identical thereto. In another embodiment of the invention, fragments ofthe antibodies having binding specificity to PACAP comprise, oralternatively consist of, one, two, three, or more, including all of thefollowing antibody fragments: the variable heavy chain region of SEQ IDNO: 402; the variable light chain region of SEQ ID NO: 422; theframework regions (SEQ ID NO: 403; SEQ ID NO: 405; SEQ ID NO: 407; andSEQ ID NO: 409) of the variable heavy chain region of SEQ ID NO: 402;and the framework regions (SEQ ID NO: 423; SEQ ID NO: 425; SEQ ID NO:427; and SEQ ID NO: 429) of the variable light chain region of SEQ IDNO: 422, or sequences that are at least 90% or 95% identical thereto.

In another embodiment of the invention, the anti-PACAP antibody is Ab10,comprising, or alternatively consisting of, SEQ ID NO: 401 and SEQ IDNO: 421, or SEQ ID NO: 402 and SEQ ID NO: 422, or an antibody orantigen-binding fragment comprising the CDRs of Ab10 and having at leastone of the biological activities set forth herein, or is an anti-PACAPantibody that competes with Ab10 in binding PACAP, preferably onecontaining sequences that are at least 90%, 95%, 96%, 97%, 98%, or 99%identical to that of Ab10, or an antibody that binds to the same oroverlapping epitope(s) on PACAP as Ab10.

In a further embodiment of the invention, antigen-binding fragmentscomprise, or alternatively consist of, Fab fragments having bindingspecificity for PACAP. With respect to antibody Ab10, the Fab fragmentpreferably includes the variable heavy chain sequence of SEQ ID NO: 402and the variable light chain sequence of SEQ ID NO: 422, or sequencesthat are at least 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.This embodiment of the invention further includes Fabs containingadditions, deletions, and variants of SEQ ID NO: 402 and/or SEQ ID NO:422 that retain the binding specificity for PACAP.

In one embodiment of the invention described herein, Fab fragments maybe produced by enzymatic digestion (e.g., papain) of Ab10. In anotherembodiment of the invention, anti-PACAP antibodies such as Ab10 and Fabfragments may be produced via expression in mammalian cells, such asCHO, NSO, or HEK 293 cells, fungal, insect, or microbial systems, suchas yeast cells (for example haploid or diploid yeast, such as haploid ordiploid Pichia) and other yeast strains. Suitable Pichia speciesinclude, but are not limited to, Pichia pastoris.

In an additional embodiment, the invention is further directed topolynucleotides encoding antibody polypeptides having bindingspecificity to PACAP, including the heavy and/or light chains of Ab10,as well as fragments, variants, and combinations of one or more of theFRs, CDRs, the variable heavy chain and variable light chain sequences,and the heavy chain and light chain sequences set forth above, includingall of them, or sequences that are at least 90% or 95% identicalthereto.

Antibody Ab20

In one embodiment, the invention includes antibodies and antigen-bindingfragments having binding specificity to PACAP that possess a heavy chainsequence comprising the sequence of SEQ ID NO: 441 which consists of theheavy chain variable region of SEQ ID NO: 442 linked to the heavy chainconstant region of SEQ ID NO: 450.

In one embodiment, the invention includes antibodies and antigen-bindingfragments having binding specificity to PACAP that contain a variableheavy chain sequence comprising the sequence set forth below:

(SEQ ID NO: 442) QSVEESGGRLVTPGTPLTLTCTVSGIDLSSYYMSWVRQAPGKGLEWIGFIDTDGSAYYATWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARDLDLWG PGTLVTVSS.

In another embodiment, the invention includes antibodies andantigen-binding fragments having binding specificity to PACAP that bindthe same epitope as Ab20, and that contain a constant heavy chainsequence comprising the polypeptide of SEQ ID NO: 1244, 1245, or 1246,or comprising the sequence set forth below:

(SEQ ID NO: 450) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In another embodiment, the invention includes antibodies andantigen-binding fragments having binding specificity to PACAP thatcontain a light chain sequence comprising the sequence of SEQ ID NO: 461which consists of the light chain variable region of SEQ ID NO: 462linked to the light chain constant region of SEQ ID NO: 470.

In another embodiment, the invention includes antibodies andantigen-binding fragments having binding specificity to PACAP thatcontain a variable light chain sequence comprising the sequence setforth below:

(SEQ ID NO: 462) AAVLTQTPSPVSAAVGGTVSISCQSSESVYSNYLAWFQQKPGQPPKFLIYEASKLASGVPSRFKGSGSGTQFTLTISDVQCDDAGTYYCAGGYSSEGVAF GGGTEVVVKR.

In another embodiment, the invention includes antibodies andantigen-binding fragments having binding specificity to PACAP, that bindthe same epitope as Ab20, and that contain a constant light chainsequence comprising the sequence set forth below:

(SEQ ID NO: 470) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC.

In another embodiment, the invention includes antibodies andantigen-binding fragments having binding specificity to PACAP thatcontain one, two, or three of the polypeptide sequences of SEQ ID NO:444; SEQ ID NO: 446; and SEQ ID NO: 448, which correspond to the CDRs(hypervariable regions) of the heavy chain sequence of SEQ ID NO: 441,or which contain the variable heavy chain sequence of SEQ ID NO: 442,and/or which further contain one, two, or three of the polypeptidesequences of SEQ ID NO: 464; SEQ ID NO: 466; and SEQ ID NO: 468, whichcorrespond to the CDRs (hypervariable regions) of the light chainsequence of SEQ ID NO: 461, or which contain the variable light chainsequence of SEQ ID NO: 462, or antibodies or antigen-binding fragmentscontaining combinations of sequences that are at least 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99% identical thereto. In another embodiment ofthe invention, the antibodies of the invention and antigen-bindingfragments comprise, or alternatively consist of, combinations of one ormore of the exemplified variable heavy chain and variable light chainsequences, or the heavy chain and light chain sequences set forth above,or sequences that are at least 90% or 95% identical thereto.

The invention further contemplates anti-PACAP antibodies andantigen-binding fragments comprising one, two, three, or four of thepolypeptide sequences of SEQ ID NO: 443; SEQ ID NO: 445; SEQ ID NO: 447;and SEQ ID NO: 449, which correspond to the FRs (constant regions) ofthe heavy chain sequence of SEQ ID NO: 441, or the variable heavy chainsequence of SEQ ID NO: 442, and/or one, two, three, or four of thepolypeptide sequences of SEQ ID NO: 463; SEQ ID NO: 465; SEQ ID NO: 467;and SEQ ID NO: 469, which correspond to the FRs (constant regions) ofthe light chain sequence of SEQ ID NO: 461, or the variable light chainsequence of SEQ ID NO: 462, or combinations of these polypeptidesequences, or sequences that are at least 80%, 90%, 95%, 96%, 97%, 98%,or 99% identical therewith.

In another embodiment of the invention, the anti-PACAP antibodies andantigen-binding fragments of the invention or fragments comprise, oralternatively consist of, combinations of one or more of the FRs, CDRs,the variable heavy chain and variable light chain sequences, and theheavy chain and light chain sequences set forth above, including all ofthem, or sequences that are at least 90% or 95% identical thereto.

In another embodiment of the invention, the anti-PACAP antibodies andantigen-binding fragments of the invention comprise, or alternativelyconsist of, the polypeptide sequence of SEQ ID NO: 441, or SEQ ID NO:442, or polypeptides that are at least 90% or 95% identical thereto. Inanother embodiment of the invention, the antibodies and antigen-bindingfragments of the invention comprise, or alternatively consist of, thepolypeptide sequence of SEQ ID NO: 461, or SEQ ID NO: 462, orpolypeptides that are at least 90% or 95% identical thereto.

In a further embodiment of the invention, the antibodies andantigen-binding fragments having binding specificity to PACAP comprise,or alternatively consist of, one, two, or three of the polypeptidesequences of SEQ ID NO: 444; SEQ ID NO: 446; and SEQ ID NO: 448, whichcorrespond to the CDRs (hypervariable regions) of the heavy chainsequence of SEQ ID NO: 441, or the variable heavy chain sequence of SEQID NO: 442, or sequences that are at least 90% or 95% identical thereto.

In a further embodiment of the invention, the antibodies andantigen-binding fragments having binding specificity to PACAP comprise,or alternatively consist of, one, two, or three of the polypeptidesequences of SEQ ID NO: 464; SEQ ID NO: 466; and SEQ ID NO: 468, whichcorrespond to the CDRs (hypervariable regions) of the light chainsequence of SEQ ID NO: 461, or the variable light chain sequence of SEQID NO: 462, or sequences that are at least 90% or 95% identical thereto.

In a further embodiment of the invention, the antibodies andantigen-binding fragments having binding specificity to PACAP comprise,or alternatively consist of, one, two, three, or four of the polypeptidesequences of SEQ ID NO: 443; SEQ ID NO: 445; SEQ ID NO: 447; and SEQ IDNO: 449, which correspond to the FRs (constant regions) of the heavychain sequence of SEQ ID NO: 441, or the variable heavy chain sequenceof SEQ ID NO: 442, or sequences that are at least 90% or 95% identicalthereto.

In a further embodiment of the invention, the subject antibodies andantigen-binding fragments having binding specificity to PACAP comprise,or alternatively consist of, one, two, three, or four of the polypeptidesequences of SEQ ID NO: 463; SEQ ID NO: 465; SEQ ID NO: 467; and SEQ IDNO: 469, which correspond to the FRs (constant regions) of the lightchain sequence of SEQ ID NO: 461, or the variable light chain sequenceof SEQ ID NO: 462, or sequences that are at least 90% or 95% identicalthereto.

The invention also contemplates anti-PACAP antibodies andantigen-binding fragments that include one or more of the antibodyfragments described herein. In one embodiment of the invention,antibodies and antigen-binding fragments having binding specificity toPACAP comprise, or alternatively consist of, one, two, three, or more,including all of the following antibody fragments: the variable heavychain region of SEQ ID NO: 442; the variable light chain region of SEQID NO: 462; the complementarity determining regions (SEQ ID NO: 444; SEQID NO: 446; and SEQ ID NO: 448) of the variable heavy chain region ofSEQ ID NO: 442; and the complementarity determining regions (SEQ ID NO:464; SEQ ID NO: 466; and SEQ ID NO: 468) of the variable light chainregion of SEQ ID NO: 462, or sequences that are at least 90% or 95%identical thereto. In another embodiment of the invention, fragments ofthe antibodies having binding specificity to PACAP comprise, oralternatively consist of, one, two, three, or more, including all of thefollowing antibody fragments: the variable heavy chain region of SEQ IDNO: 442; the variable light chain region of SEQ ID NO: 462; theframework regions (SEQ ID NO: 443; SEQ ID NO: 445; SEQ ID NO: 447; andSEQ ID NO: 449) of the variable heavy chain region of SEQ ID NO: 442;and the framework regions (SEQ ID NO: 463; SEQ ID NO: 465; SEQ ID NO:467; and SEQ ID NO: 469) of the variable light chain region of SEQ IDNO: 462, or sequences that are at least 90% or 95% identical thereto.

In another embodiment of the invention, the anti-PACAP antibody is Ab20,comprising, or alternatively consisting of, SEQ ID NO: 441 and SEQ IDNO: 461, or SEQ ID NO: 442 and SEQ ID NO: 462, or an antibody orantigen-binding fragment comprising the CDRs of Ab20 and having at leastone of the biological activities set forth herein, or is an anti-PACAPantibody that competes with Ab20 in binding PACAP, preferably onecontaining sequences that are at least 90%, 95%, 96%, 97%, 98%, or 99%identical to that of Ab20, or an antibody that binds to the same oroverlapping epitope(s) on PACAP as Ab20.

In a further embodiment of the invention, antigen-binding fragmentscomprise, or alternatively consist of, Fab fragments having bindingspecificity for PACAP. With respect to antibody Ab20, the Fab fragmentpreferably includes the variable heavy chain sequence of SEQ ID NO: 442and the variable light chain sequence of SEQ ID NO: 462, or sequencesthat are at least 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.This embodiment of the invention further includes Fabs containingadditions, deletions, and variants of SEQ ID NO: 442 and/or SEQ ID NO:462 that retain the binding specificity for PACAP.

In one embodiment of the invention described herein, Fab fragments maybe produced by enzymatic digestion (e.g., papain) of Ab20. In anotherembodiment of the invention, anti-PACAP antibodies such as Ab20 and Fabfragments may be produced via expression in mammalian cells, such asCHO, NSO, or HEK 293 cells, fungal, insect, or microbial systems, suchas yeast cells (for example haploid or diploid yeast, such as haploid ordiploid Pichia) and other yeast strains. Suitable Pichia speciesinclude, but are not limited to, Pichia pastoris.

In an additional embodiment, the invention is further directed topolynucleotides encoding antibody polypeptides having bindingspecificity to PACAP, including the heavy and/or light chains of Ab20,as well as fragments, variants, and combinations of one or more of theFRs, CDRs, the variable heavy chain and variable light chain sequences,and the heavy chain and light chain sequences set forth above, includingall of them, or sequences that are at least 90% or 95% identicalthereto.

In another embodiment, the invention contemplates an isolated anti-PACAPantibody comprising a V_(H) polypeptide sequence selected from: SEQ IDNO: 402, SEQ ID NO: 442, or a variant thereof; and further comprising aV_(L) polypeptide sequence selected from: SEQ ID NO: 422, SEQ ID NO:462, or a variant thereof, wherein one or more of the framework regionresidues (“FR residues”) and/or CDR residues in said V_(H) or V_(L)polypeptide has been substituted with another amino acid residueresulting in an anti-PACAP antibody that specifically binds PACAP. Theinvention also includes humanized and chimeric forms of theseantibodies. The chimeric and humanized antibodies may include an Fcderived from IgG1, IgG2, IgG3, or IgG4 constant regions.

In one embodiment of the invention, the chimeric or humanized antibodiesor fragments or V_(H) or V_(L) polypeptides originate or are derivedfrom one or more rabbit antibodies, e.g., a rabbit antibody isolatedfrom a clonal rabbit B cell population.

In some aspects, the invention provides a vector comprising a nucleicacid molecule encoding an anti-PACAP antibody or fragment thereof asdisclosed herein. In some embodiments, the invention provides a hostcell comprising a nucleic acid molecule encoding an anti-PACAP antibodyor fragment thereof as disclosed herein.

In some aspects, the invention provides an isolated antibody or antigenbinding fragment thereof that competes for binding to PACAP with anantibody or antigen binding fragment thereof disclosed herein.

In some aspects, the invention provides a nucleic acid molecule encodingan antibody or antigen binding fragment thereof as disclosed herein.

In some aspects, the invention provides a pharmaceutical or diagnosticcomposition comprising at least one antibody or antigen binding fragmentthereof as disclosed herein.

In some aspects, the invention provides a method for treating orpreventing a condition associated with elevated PACAP levels in asubject, comprising administering to a subject in need thereof aneffective amount of at least one isolated antibody or antigen bindingfragment thereof as disclosed herein.

In some aspects, the invention provides a method of inhibiting bindingof PACAP to PAC1-R, VPAC1-R, and/or VPAC2-R in a subject comprisingadministering an effective amount of at least one antibody or antigenbinding fragment thereof as disclosed herein.

In some aspects, the invention provides an antibody or antigen bindingfragment thereof that selectively binds to PACAP, wherein the antibodyor antigen binding fragment thereof binds to PACAP with a K_(D) of lessthan or equal to 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M,5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, or 10⁻¹³ M; preferably, with a K_(D)of less than or equal to 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹²M, or 10⁻¹² M; more preferably, with a K_(D) that is less than about 100pM, less than about 50 pM, less than about 40 pM, less than about 25 pM,less than about 1 pM, between about 10 pM and about 100 pM, betweenabout 1 pM and about 100 pM, or between about 1 pM and about 10 pM.Preferably, the anti-PACAP antibody or antigen binding fragment thereofhas no cross-reactivity or minimal cross-reactivity with VIP.

The inventive antibodies and antigen binding fragments thereof may bemodified post-translationally to add effector moieties such as chemicallinkers, detectable moieties such as for example fluorescent dyes,enzymes, substrates, bioluminescent materials, radioactive materials,and chemiluminescent moieties, or functional moieties such as forexample streptavidin, avidin, biotin, a cytotoxin, a cytotoxic agent,and radioactive materials.

Antibodies and antigen binding fragments thereof may also be chemicallymodified to provide additional advantages such as increased solubility,stability and circulating time (in vivo half-life) of the polypeptide,or decreased immunogenicity (See U.S. Pat. No. 4,179,337). The chemicalmoieties for derivatization may be selected from water soluble polymerssuch as polyethylene glycol, ethylene glycol/propylene glycolcopolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, and thelike. The antibodies and fragments thereof may be modified at randompositions within the molecule, or at predetermined positions within themolecule and may include one, two, three, or more attached chemicalmoieties.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 1 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog). For example,the polyethylene glycol may have an average molecular weight of about200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000,11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500,16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000,25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000,75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa. Branchedpolyethylene glycols are described, for example, in U.S. Pat. No.5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol., 56:59-72 (1996);Vorobjev et al., Nucleosides and Nucleotides, 18:2745-2750 (1999); andCaliceti et al., Bioconjug. Chem., 10:638-646 (1999), the disclosures ofeach of which are incorporated herein by reference.

There are a number of attachment methods available to those skilled inthe art (See e.g., EP 0 401 384, herein incorporated by reference,disclosing a method of coupling PEG to G-CSF; and Malik et al., Exp.Hematol., 20:1028-1035 (1992) (reporting pegylation of GM-CSF usingtresyl chloride)). For example, polyethylene glycol may be covalentlybound through amino acid residues via a reactive group, such as, a freeamino or carboxyl group. Reactive groups are those to which an activatedpolyethylene glycol molecule may be bound. The amino acid residueshaving a free amino group may include lysine residues and the N-terminalamino acid residues; those having a free carboxyl group may includeaspartic acid residues glutamic acid residues and the C-terminal aminoacid residue. Sulfhydryl groups may also be used as a reactive group forattaching the polyethylene glycol molecules. Preferred for therapeuticpurposes is attachment at an amino group, such as attachment at theN-terminus or lysine group.

As suggested above, polyethylene glycol may be attached to proteins vialinkage to any of a number of amino acid residues. For example,polyethylene glycol can be linked to polypeptides via covalent bonds tolysine, histidine, aspartic acid, glutamic acid, or cysteine residues.One or more reaction chemistries may be employed to attach polyethyleneglycol to specific amino acid residues (e.g., lysine, histidine,aspartic acid, glutamic acid, or cysteine) or to more than one type ofamino acid residue (e.g., lysine, histidine, aspartic acid, glutamicacid, cysteine and combinations thereof).

Alternatively, antibodies or antigen binding fragments thereof may haveincreased in vivo half-lives via fusion with albumin (including but notlimited to recombinant human serum albumin or fragments or variantsthereof (See, e.g., U.S. Pat. No. 5,876,969, EP 0 413 622, and U.S. Pat.No. 5,766,883, herein incorporated by reference in their entirety)), orother circulating blood proteins such as transferrin or ferritin. In apreferred embodiment, polypeptides and/or antibodies of the presentinvention (including fragments or variants thereof) are fused with themature form of human serum albumin (i.e., amino acids 1-585 of humanserum albumin as shown in FIGS. 1 and 2 of EP 0 322 094) which is hereinincorporated by reference in its entirety. Polynucleotides encodingfusion proteins of the invention are also encompassed by the invention.

Regarding detectable moieties, further exemplary enzymes include, butare not limited to, horseradish peroxidase, acetylcholinesterase,alkaline phosphatase, beta-galactosidase, and luciferase. Furtherexemplary fluorescent materials include, but are not limited to,rhodamine, fluorescein, fluorescein isothiocyanate, umbelliferone,dichlorotriazinylamine, phycoerythrin, and dansyl chloride. Furtherexemplary chemiluminescent moieties include, but are not limited to,luminol. Further exemplary bioluminescent materials include, but are notlimited to, luciferin and aequorin. Further exemplary radioactivematerials include, but are not limited to, Iodine 125 (¹²⁵I), Carbon 14(¹⁴C), Sulfur 35 (³⁵S), Tritium (³H) and Phosphorus 32 (³²P).

Methods are known in the art for conjugating an antibody or antigenbinding fragment thereof to a detectable moiety and the like, such asfor example those methods described by Hunter et al., Nature, 144:945(1962); David et al., Biochemistry, 13:1014 (1974); Pain et al., J.Immunol. Meth., 40:219 (1981); and Nygren, J., Histochem. and Cytochem.,30:407 (1982).

Embodiments described herein further include variants and equivalentsthat are substantially homologous to the antibodies, antibody fragments,diabodies, SMIPs, camelbodies, nanobodies, IgNAR, polypeptides, variableregions, and CDRs set forth herein. These may contain, e.g.,conservative substitution mutations, (i.e., the substitution of one ormore amino acids by similar amino acids). For example, conservativesubstitution refers to the substitution of an amino acid with anotherwithin the same general class, e.g., one acidic amino acid with anotheracidic amino acid, one basic amino acid with another basic amino acid,or one neutral amino acid by another neutral amino acid. What isintended by a conservative amino acid substitution is well known in theart.

In another embodiment, the invention contemplates polypeptide sequenceshaving at least 90% or greater sequence homology to any one or more ofthe polypeptide sequences of antigen binding fragments, variable regionsand CDRs set forth herein. More preferably, the invention contemplatespolypeptide sequences having at least 95% or greater sequence homology,even more preferably at least 98% or greater sequence homology, andstill more preferably at least 99% or greater sequence homology to anyone or more of the polypeptide sequences of antigen binding fragments,variable regions, and CDRs set forth herein.

Methods for determining homology between nucleic acid and amino acidsequences are well known to those of ordinary skill in the art.

In another embodiment, the invention further contemplates theabove-recited polypeptide homologs of the antigen binding fragments,variable regions and CDRs set forth herein further having anti-PACAPactivity. Non-limiting examples of anti-PACAP activity are set forthherein, e.g., ability to inhibit PACAP binding to PAC1-R, VPAC1-R,and/or VPAC2-R, thereby resulting in the reduced production of cAMP.

In another embodiment, the invention further contemplates the generationand use of antibodies that bind any of the foregoing sequences,including, but not limited to, anti-idiotypic antibodies. In anexemplary embodiment, such an anti-idiotypic antibody could beadministered to a subject who has received an anti-PACAP antibody tomodulate, reduce, or neutralize, the effect of the anti-PACAP antibody.Such antibodies could also be useful for treatment of an autoimmunedisease characterized by the presence of anti-PACAP antibodies. Afurther exemplary use of such antibodies, e.g., anti-idiotypicantibodies, is for detection of the anti-PACAP antibodies of the presentinvention, for example to monitor the levels of the anti-PACAPantibodies present in a subject's blood or other bodily fluids. Forexample, in one embodiment, the invention provides a method of using theanti-idiotypic antibody to monitor the in vivo levels of said anti-PACAPantibody or antigen binding fragment thereof in a subject or toneutralize said anti-PACAP antibody in a subject being administered saidanti-PACAP antibody or antigen binding fragment thereof.

The present invention also contemplates anti-PACAP antibodies comprisingany of the polypeptide or polynucleotide sequences described hereinsubstituted for any of the other polynucleotide sequences describedherein. For example, without limitation thereto, the present inventioncontemplates antibodies comprising the combination of any of thevariable light chain and variable heavy chain sequences describedherein, and further contemplates antibodies resulting from substitutionof any of the CDR sequences described herein for any of the other CDRsequences described herein.

Exemplary Polynucleotides Encoding Anti-PACAP Antibody Polypeptides

The invention is further directed to polynucleotides encoding antibodypolypeptides having binding specificity to PACAP.

Antibody Ab10

In one embodiment, the invention is further directed to polynucleotidesencoding antibody polypeptides having binding specificity to PACAP. Inone embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the polynucleotide sequence ofSEQ ID NO: 411 which encodes the heavy chain sequence of SEQ ID NO: 401and which consists of the heavy chain variable region coding sequence ofSEQ ID NO: 412 and the heavy chain constant region coding sequence ofSEQ ID NO: 420.

In another embodiment of the invention, the polynucleotides of theinvention comprise, or alternatively consist of, the followingpolynucleotide sequence encoding the variable heavy chain polypeptidesequence of SEQ ID NO: 402:

(SEQ ID NO: 412) cagtcggtggaggagtccgggggtcgcctggtcacgcctgggacacccctgacactcacctgcacagtctctggaatcgacctcaatagctactacatgacctgggtccgccaggctccagggaaggggctggaatggatcggattcattgatgctggtggtgacgcatactacgcgagctgggcgaaaggccgattcaccatctccaaaacctcgaccacggtggatctgaaaatcaccagtccgacaaccgaggacacggccacctatttctgtgccagagatcttgacttgtggggccagggcaccctggtcaccgtctcgagc.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the constant heavy chain polypeptide sequence of SEQID NO: 410:

(SEQ ID NO: 420) gcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacgcgagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa.

In another embodiment of the invention, polynucleotides comprise, oralternatively consist of, the polynucleotide sequence of SEQ ID NO: 431which encodes the light chain polypeptide sequence of SEQ ID NO: 421 andwhich consists of the light chain variable region coding sequence of SEQID NO: 432 and the light chain constant region coding sequence of SEQ IDNO: 440.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the variable light chain polypeptide sequence of SEQID NO: 422:

(SEQ ID NO: 432) gccgccgtgctgacccagactccatctcccgtgtctgcagctgtgggaggcacagtcaccatcaattgccagtccagtgagagtgtttacggtaactacttagcctggtttcagcagaaaccagggcagcctcccaagctcctgatctacgaagcatccaaactggaatctggggtcccatcgcggttcagcggcagtggatctgggacacagttcactctcaccatcagcgacttgcagtgtgacgatgctgccacttactactgtgcaggcggtgatattagtgaaggtgttgctttcggcggagggaccgaggtggtggtcaaacgt.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the constant light chain polypeptide sequence of SEQID NO: 430:

(SEQ ID NO: 440) acggtagcggccccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagc ttcaacaggggagagtgt.

In a further embodiment of the invention, polynucleotides encodingantigen-binding fragments having binding specificity to PACAP comprise,or alternatively consist of, one or more of the polynucleotide sequencesof SEQ ID NO: 414; SEQ ID NO: 416; and SEQ ID NO: 418, which correspondto polynucleotides encoding the CDRs (hypervariable regions) of theheavy chain sequence of SEQ ID NO: 401, or the variable heavy chainsequence of SEQ ID NO: 402, and/or one or more of the polynucleotidesequences of SEQ ID NO: 434; SEQ ID NO: 436; and SEQ ID NO: 438, whichcorrespond to the CDRs (hypervariable regions) of the light chainsequence of SEQ ID NO: 421, or the variable light chain sequence of SEQID NO: 422, or combinations of these polynucleotide sequences. Inanother embodiment of the invention, the polynucleotides encoding theantibodies of the invention or antigen-binding fragments thereofcomprise, or alternatively consist of, combinations of polynucleotidesencoding one or more of the CDRs, the variable heavy chain and variablelight chain sequences, and the heavy chain and light chain sequences setforth above, including all of them.

In a further embodiment of the invention, polynucleotides encodingantigen-binding fragments having binding specificity to PACAP comprise,or alternatively consist of, one or more of the polynucleotide sequencesof SEQ ID NO: 413; SEQ ID NO: 415; SEQ ID NO: 417; and SEQ ID NO: 419,which correspond to polynucleotides encoding the FRs (constant regions)of the heavy chain sequence of SEQ ID NO: 401, or the variable heavychain sequence of SEQ ID NO: 402, and/or one or more of thepolynucleotide sequences of SEQ ID NO: 433; SEQ ID NO: 435; SEQ ID NO:437; and SEQ ID NO: 439, which correspond to the FRs (constant regions)of the light chain sequence of SEQ ID NO: 421, or the variable lightchain sequence of SEQ ID NO: 422, or combinations of thesepolynucleotide sequences. In another embodiment of the invention, thepolynucleotides encoding the antibodies of the invention or fragmentsthereof comprise, or alternatively consist of, combinations of one ormore of the FRs, the variable heavy chain and variable light chainsequences, and the heavy chain and light chain sequences set forthabove, including all of them.

The invention also contemplates polynucleotide sequences including oneor more of the polynucleotide sequences encoding antigen-bindingfragments described herein. In one embodiment of the invention,polynucleotides encoding antigen-binding fragments having bindingspecificity to PACAP comprise, or alternatively consist of, one, two,three or more, including all of the following polynucleotides encodingantigen-binding fragments: the polynucleotide SEQ ID NO: 411 encodingthe heavy chain sequence of SEQ ID NO: 401; the polynucleotide SEQ IDNO: 412 encoding the variable heavy chain sequence of SEQ ID NO: 402;the polynucleotide SEQ ID NO: 431 encoding the light chain sequence ofSEQ ID NO: 421; the polynucleotide SEQ ID NO: 432 encoding the variablelight chain sequence of SEQ ID NO: 422; polynucleotides encoding theCDRs (SEQ ID NO: 414; SEQ ID NO: 416; and SEQ ID NO: 418) of the heavychain sequence of SEQ ID NO: 401, or the variable heavy chain sequenceof SEQ ID NO: 402; polynucleotides encoding the CDRs (SEQ ID NO: 434;SEQ ID NO: 436; and SEQ ID NO: 438) of the light chain sequence of SEQID NO: 421, or the variable light chain sequence of SEQ ID NO: 422;polynucleotides encoding the FRs (SEQ ID NO: 413; SEQ ID NO: 415; SEQ IDNO: 417; and SEQ ID NO: 419) of the heavy chain sequence of SEQ ID NO:401, or the variable heavy chain sequence of SEQ ID NO: 402; andpolynucleotides encoding the FRs (SEQ ID NO: 433; SEQ ID NO: 435; SEQ IDNO: 437; and SEQ ID NO: 439) of the light chain sequence of SEQ ID NO:421, or the variable light chain sequence of SEQ ID NO: 422.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, polynucleotides encoding Fabfragments having binding specificity for PACAP. With respect to antibodyAb10, the polynucleotides encoding the full length Ab10 antibodycomprise, or alternatively consist of, the polynucleotide SEQ ID NO: 411encoding the heavy chain sequence of SEQ ID NO: 401, and thepolynucleotide SEQ ID NO: 431 encoding the light chain sequence of SEQID NO: 421.

Another embodiment of the invention contemplates these polynucleotidesincorporated into an expression vector for expression in mammalian cellssuch as CHO, NSO, or HEK-293 cells, or in fungal, insect, or microbialsystems such as yeast cells such as the yeast Pichia. Suitable Pichiaspecies include, but are not limited to, Pichia pastoris. In oneembodiment of the invention described herein, Fab fragments can beproduced by enzymatic digestion (e.g., papain) of Ab10 followingexpression of the full-length polynucleotides in a suitable host. Inanother embodiment of the invention, anti-PACAP antibodies, such as Ab10or Fab fragments thereof, can be produced via expression of Ab10polynucleotides in mammalian cells such as CHO, NSO, or HEK 293 cells,fungal, insect, or microbial systems such as yeast cells (for examplediploid yeast such as diploid Pichia) and other yeast strains. SuitablePichia species include, but are not limited to, Pichia pastoris.

Antibody Ab20

In one embodiment, the invention is further directed to polynucleotidesencoding antibody polypeptides having binding specificity to PACAP. Inone embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the polynucleotide sequence ofSEQ ID NO: 451 which encodes the heavy chain sequence of SEQ ID NO: 441and which consists of the heavy chain variable region coding sequence ofSEQ ID NO: 452 and the heavy chain constant region coding sequence ofSEQ ID NO: 460.

In another embodiment of the invention, the polynucleotides of theinvention comprise, or alternatively consist of, the followingpolynucleotide sequence encoding the variable heavy chain polypeptidesequence of SEQ ID NO: 442:

(SEQ ID NO: 452) cagtcggtggaggagtccgggggtcgcctggtcacgcctgggacacccctgacactcacctgcacagtctctggaatcgacctcagtagctactacatgagctgggtccgccaggctccagggaaggggctggaatggatcggattcattgatactgatggtagcgcatactacgcgacctgggcgaaaggccgattcaccatctccaaaacctcgaccacggtggatctgaaaatcaccagtccgacaaccgaggacacggccacctatttctgtgccagagatcttgacttgtggggcccgggcaccctcgtcaccgtctcgagc.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the constant heavy chain polypeptide sequence of SEQID NO: 450:

(SCQ ID NO: 460) gcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa.

In another embodiment of the invention, polynucleotides comprise, oralternatively consist of, the polynucleotide sequence of SEQ ID NO: 471which encodes the light chain polypeptide sequence of SEQ ID NO: 461 andwhich consists of the light chain variable region coding sequence of SEQID NO: 472 and the light chain constant region coding sequence of SEQ IDNO: 480.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the variable light chain polypeptide sequence of SEQID NO: 462:

(SEQ ID NO: 472) gccgccgtgctgacccagactccatctcccgtgtagcagagtgggaggcacagtcagcatcagttgccagtccagtgagagtgatatagtaactacttagcctggtttcagcagaaaccagggcagcctcctaagttcttgatctacgaagcatccaaactggcatctggggtcccatcgcggttcaaaggcagtggatctgggacacagttcactctcaccatcagcgacgtgcagtgtgacgatgctggcacttactactgtgcaggcggctatagtagtgaaggtgttgctttcggcggagggaccgaggtggtggtcaaacgt.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the constant light chain polypeptide sequence of SEQID NO: 470:

(SEQ ID NO: 480) acggtagcggccccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagc ttcaacaggggagagtgt.

In a further embodiment of the invention, polynucleotides encodingantigen-binding fragments having binding specificity to PACAP comprise,or alternatively consist of, one or more of the polynucleotide sequencesof SEQ ID NO: 454; SEQ ID NO: 456; and SEQ ID NO: 458, which correspondto polynucleotides encoding the CDRs (hypervariable regions) of theheavy chain sequence of SEQ ID NO: 441, or the variable heavy chainsequence of SEQ ID NO: 442, and/or one or more of the polynucleotidesequences of SEQ ID NO: 474; SEQ ID NO: 476; and SEQ ID NO: 478, whichcorrespond to the CDRs (hypervariable regions) of the light chainsequence of SEQ ID NO: 461, or the variable light chain sequence of SEQID NO: 462, or combinations of these polynucleotide sequences. Inanother embodiment of the invention, the polynucleotides encoding theantibodies of the invention or antigen-binding fragments thereofcomprise, or alternatively consist of, combinations of polynucleotidesencoding one or more of the CDRs, the variable heavy chain and variablelight chain sequences, and the heavy chain and light chain sequences setforth above, including all of them.

In a further embodiment of the invention, polynucleotides encodingantigen-binding fragments having binding specificity to PACAP comprise,or alternatively consist of, one or more of the polynucleotide sequencesof SEQ ID NO: 453; SEQ ID NO: 455; SEQ ID NO: 457; and SEQ ID NO: 459,which correspond to polynucleotides encoding the FRs (constant regions)of the heavy chain sequence of SEQ ID NO: 441, or the variable heavychain sequence of SEQ ID NO: 442, and/or one or more of thepolynucleotide sequences of SEQ ID NO: 473; SEQ ID NO: 475; SEQ ID NO:477; and SEQ ID NO: 479, which correspond to the FRs (constant regions)of the light chain sequence of SEQ ID NO: 461, or the variable lightchain sequence of SEQ ID NO: 462, or combinations of thesepolynucleotide sequences. In another embodiment of the invention, thepolynucleotides encoding the antibodies of the invention or fragmentsthereof comprise, or alternatively consist of, combinations of one ormore of the FRs, the variable heavy chain and variable light chainsequences, and the heavy chain and light chain sequences set forthabove, including all of them.

The invention also contemplates polynucleotide sequences including oneor more of the polynucleotide sequences encoding antigen-bindingfragments described herein. In one embodiment of the invention,polynucleotides encoding antigen-binding fragments having bindingspecificity to PACAP comprise, or alternatively consist of, one, two,three or more, including all of the following polynucleotides encodingantigen-binding fragments: the polynucleotide SEQ ID NO: 451 encodingthe heavy chain sequence of SEQ ID NO: 441; the polynucleotide SEQ IDNO: 452 encoding the variable heavy chain sequence of SEQ ID NO: 442;the polynucleotide SEQ ID NO: 471 encoding the light chain sequence ofSEQ ID NO: 461; the polynucleotide SEQ ID NO: 472 encoding the variablelight chain sequence of SEQ ID NO: 462; polynucleotides encoding theCDRs (SEQ ID NO: 454; SEQ ID NO: 456; and SEQ ID NO: 458) of the heavychain sequence of SEQ ID NO: 441, or the variable heavy chain sequenceof SEQ ID NO: 442; polynucleotides encoding the CDRs (SEQ ID NO: 474;SEQ ID NO: 476; and SEQ ID NO: 478) of the light chain sequence of SEQID NO: 461, or the variable light chain sequence of SEQ ID NO: 462;polynucleotides encoding the FRs (SEQ ID NO: 453; SEQ ID NO: 455; SEQ IDNO: 457; and SEQ ID NO: 459) of the heavy chain sequence of SEQ ID NO:441, or the variable heavy chain sequence of SEQ ID NO: 442; andpolynucleotides encoding the FRs (SEQ ID NO: 473; SEQ ID NO: 475; SEQ IDNO: 477; and SEQ ID NO: 479) of the light chain sequence of SEQ ID NO:461, or the variable light chain sequence of SEQ ID NO: 462.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, polynucleotides encoding Fabfragments having binding specificity for PACAP. With respect to antibodyAb20, the polynucleotides encoding the full length Ab20 antibodycomprise, or alternatively consist of, the polynucleotide SEQ ID NO: 451encoding the heavy chain sequence of SEQ ID NO: 441, and thepolynucleotide SEQ ID NO: 471 encoding the light chain sequence of SEQID NO: 461.

Another embodiment of the invention contemplates these polynucleotidesincorporated into an expression vector for expression in mammalian cellssuch as CHO, NSO, or HEK-293 cells, or in fungal, insect, or microbialsystems such as yeast cells such as the yeast Pichia. Suitable Pichiaspecies include, but are not limited to, Pichia pastoris. In oneembodiment of the invention described herein, Fab fragments can beproduced by enzymatic digestion (e.g., papain) of Ab20 followingexpression of the full-length polynucleotides in a suitable host. Inanother embodiment of the invention, anti-PACAP antibodies, such as Ab20or Fab fragments thereof, can be produced via expression of Ab20polynucleotides in mammalian cells such as CHO, NSO, or HEK 293 cells,fungal, insect, or microbial systems such as yeast cells (for examplediploid yeast such as diploid Pichia) and other yeast strains. SuitablePichia species include, but are not limited to, Pichia pastoris.

Host cells and vectors comprising said polynucleotides are alsocontemplated.

The invention further contemplates vectors comprising the polynucleotidesequences encoding the variable heavy and light chain polypeptidesequences, as well as the individual CDRs (hypervariable regions), asset forth herein, as well as host cells comprising said vectorsequences. In embodiments of the invention, the host cells are mammaliancells, such as CHO cells. In embodiments of the invention, the hostcells are yeast cells, such as yeast cells of the genus Pichia

B-Cell Screening and Isolation

In one embodiment, the present invention contemplates the preparationand isolation of a clonal population of antigen-specific B-cells thatmay be used for isolating at least one PACAP antigen-specific cell,which can be used to produce a monoclonal antibody against PACAP, whichis specific to a desired PACAP antigen, or a nucleic acid sequencecorresponding to such an antibody. Methods of preparing and isolatingsaid clonal population of antigen-specific B-cells are taught, forexample, in U.S. Patent Publication No. 2007/0269868 to Carvalho-Jensenet al., the disclosure of which is herein incorporated by reference inits entirety. Methods of preparing and isolating said clonal populationof antigen-specific B-cells are also taught herein in the examples.Methods of “enriching” a cell population by size or density are known inthe art (See, e.g., U.S. Pat. No. 5,627,052). These steps can be used inaddition to enriching the cell population by antigen-specificity.

Methods of Humanizing Antibodies

In another embodiment, the present invention contemplates methods forhumanizing antibody heavy and light chains. Methods for humanizingantibody heavy and light chains that may be applied to anti-PACAPantibodies are taught, for example, in U.S. Patent Publication No.2009/0022659 to Olson et al., and in U.S. Pat. No. 7,935,340 toGarcia-Martinez et al., the disclosures of each of which are hereinincorporated by reference in their entireties.

Methods of Producing Antibodies and Fragments Thereof

In another embodiment, the present invention contemplates methods forproducing anti-PACAP antibodies and fragments thereof. Methods forproducing anti-PACAP antibodies and fragments thereof secreted frompolyploidal, preferably diploid or tetraploid strains of matingcompetent yeast are taught, for example, in U.S. Patent Publication No.2009/0022659 to Olson et al., and in U.S. Pat. No. 7,935,340 toGarcia-Martinez et al., the disclosures of each of which are hereinincorporated by reference in their entireties.

Other methods of producing antibodies are well known to those ofordinary skill in the art. For example, methods of producing chimericantibodies are now well known in the art (See, for example, U.S. Pat.No. 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci.U.S.A., 81:8651-55, 1984; Neuberger et al., Nature, 314:268-270, 1985;Boulianne, G. L. et al., Nature, 312:643-46, 1984, the disclosures ofeach of which are herein incorporated by reference in their entireties).

Likewise, other methods of producing humanized antibodies are now wellknown in the art (See, for example, U.S. Pat. Nos. 5,530,101, 5,585,089,5,693,762, and 6,180,370 to Queen et al; U.S. Pat. Nos. 5,225,539 and6,548,640 to Winter; U.S. Pat. Nos. 6,054,297, 6,407,213 and 6,639,055to Carter et al; U.S. Pat. No. 6,632,927 to Adair; Jones, P. T. et al.,Nature, 321:522-525, 1986; Reichmann, L. et al., Nature, 332:323-327,1988; Verhoeyen, M. et al., Science, 239:1534-36, 1988, the disclosuresof each of which are herein incorporated by reference in theirentireties).

Antibody polypeptides of the invention having PACAP binding specificitymay also be produced by constructing, using conventional techniques wellknown to those of ordinary skill in the art, an expression vectorcontaining a promoter (optionally as a component of a eukaryotic orprokaryotic operon) and a DNA sequence encoding an antibody heavy chainin which the DNA sequence encoding the CDRs required for antibodyspecificity is derived from a non-human cell source, preferably a rabbitB-cell source, while the DNA sequence encoding the remaining parts ofthe antibody chain is derived from a human cell source.

A second expression vector is produced using the same conventional meanswell known to those of ordinary skill in the art, said expression vectorcontaining a promoter (optionally as a component of a eukaryotic orprokaryotic operon) and a DNA sequence encoding an antibody light chainin which the DNA sequence encoding the CDRs required for antibodyspecificity is derived from a non-human cell source, preferably a rabbitB-cell source, while the DNA sequence encoding the remaining parts ofthe antibody chain is derived from a human cell source.

The expression vectors are transfected into a host cell by conventiontechniques well known to those of ordinary skill in the art to produce atransfected host cell, said transfected host cell cultured byconventional techniques well known to those of ordinary skill in the artto produce said antibody polypeptides.

The host cell may be co-transfected with the two expression vectorsdescribed above, the first expression vector containing DNA encoding apromoter (optionally as a component of a eukaryotic or prokaryoticoperon) and a light chain-derived polypeptide and the second vectorcontaining DNA encoding a promoter (optionally as a component of aeukaryotic or prokaryotic operon) and a heavy chain-derived polypeptide.The two vectors contain different selectable markers, but preferablyachieve substantially equal expression of the heavy and light chainpolypeptides. Alternatively, a single vector may be used, the vectorincluding DNA encoding both the heavy and light chain polypeptides. Thecoding sequences for the heavy and light chains may comprise cDNA,genomic DNA, or both.

The host cells used to express the antibody polypeptides may be either abacterial cell such as E. coli, or a eukaryotic cell such as P.pastoris. In one embodiment of the invention, a mammalian cell of awell-defined type for this purpose, such as a myeloma cell, a CHO cellline, a NSO cell line, or a HEK293 cell line may be used.

The general methods by which the vectors may be constructed,transfection methods required to produce the host cell and culturingmethods required to produce the antibody polypeptides from said hostcells all include conventional techniques. Although preferably the cellline used to produce the antibody is a mammalian cell line, any othersuitable cell line, such as a bacterial cell line such as an E.coli-derived bacterial strain, or a yeast cell line, may alternativelybe used.

Similarly, once produced the antibody polypeptides may be purifiedaccording to standard procedures in the art, such as for examplecross-flow filtration, ammonium sulphate precipitation, affinity columnchromatography, hydrophobic interaction chromatography (“HIC”), and thelike.

The antibody polypeptides described herein may also be used for thedesign and synthesis of either peptide or non-peptide mimetics thatwould be useful for the same therapeutic applications as the antibodypolypeptides of the invention (See, for example, Saragobi et al.,Science, 253:792-795, 1991, the contents of which are hereinincorporated by reference in its entirety).

Screening Assays

The screening assays described here are designed to identify highaffinity anti-PACAP Abs which may be useful in the treatment of diseasesand disorders associated with PACAP in subjects exhibiting symptoms of aPACAP associated disease or disorder.

In some embodiments, the antibody is used as a diagnostic tool. Theantibody can be used to assay the amount of PACAP present in a sampleand/or subject. As will be appreciated by one of skill in the art, suchantibodies need not be neutralizing antibodies. In some embodiments, thediagnostic antibody is not a neutralizing antibody. In some embodiments,the diagnostic antibody binds to a different epitope than theneutralizing antibody binds to. In some embodiments, the two antibodiesdo not compete with one another.

In some embodiments, the antibodies disclosed herein are used orprovided in an assay kit and/or method for the detection of PACAP inmammalian tissues or cells in order to screen/diagnose for a disease ordisorder associated with changes in levels of PACAP. The kit comprisesan antibody that binds PACAP and means for indicating the binding of theantibody with PACAP, if present, and optionally PACAP protein levels.Various means for indicating the presence of an antibody can be used.For example, fluorophores, other molecular probes, or enzymes can belinked to the antibody and the presence of the antibody can be observedin a variety of ways. The method for screening for such disorders caninvolve the use of the kit, or simply the use of one of the disclosedantibodies and the determination of whether the antibody binds to PACAPin a sample. As will be appreciated by one of skill in the art, high orelevated levels of PACAP will result in larger amounts of the antibodybinding to PACAP in the sample. Thus, degree of antibody binding can beused to determine how much PACAP is in a sample. Subjects or sampleswith an amount of PACAP that is greater than a predetermined amount(e.g., an amount or range that a person without a PACAP-related disorderwould have) can be characterized as having a PACAP-mediated disorder,e.g., migraine, headache, pain, or other condition.

The present invention further provides for a kit for detecting bindingof an anti-PACAP antibody of the invention to PACAP. In particular, thekit may be used to detect the presence of PACAP specifically reactivewith an anti-PACAP antibody of the invention or an immunoreactivefragment thereof. The kit may also include an antibody bound to asubstrate, a secondary antibody reactive with the antigen and a reagentfor detecting a reaction of the secondary antibody with the antigen.Such a kit may be an ELISA kit and can comprise the substrate, primaryand secondary antibodies when appropriate, and any other necessaryreagents such as detectable moieties, enzyme substrates, and colorreagents, for example as described herein. The diagnostic kit may alsobe in the form of an immunoblot kit. The diagnostic kit may also be inthe form of a chemiluminescent kit (Meso Scale Discovery, Gaithersburg,Md.). The diagnostic kit may also be a lanthanide-based detection kit(PerkinElmer, San Jose, Calif.).

A skilled clinician would understand that a biological sample includes,but is not limited to, sera, plasma, urine, saliva, mucous, pleuralfluid, synovial fluid, and spinal fluid.

Methods of Ameliorating or Reducing Symptoms of or Treating, orPreventing, Diseases and Disorders Associated with PACAP

In another embodiment of the invention, anti-PACAP antibodies describedherein, or antigen binding fragments thereof, are useful forameliorating or reducing the symptoms of, or treating, or preventing,diseases and disorders associated with PACAP. Anti-PACAP antibodiesdescribed herein, or antigen binding fragments thereof, as well ascombinations, can also be administered in a therapeutically effectiveamount to patients in need of treatment of diseases and disordersassociated with PACAP in the form of a pharmaceutical composition asdescribed in greater detail below.

In another embodiment of the invention, anti-PACAP antibodies describedherein, or antigen binding fragments thereof, are useful (either aloneor in combination with another agent) for ameliorating or reducing thesymptoms of, or treating, or preventing a disease or conditionassociated with PACAP.

In another embodiment of the invention, anti-PACAP antibodies describedherein, or antigen binding fragments thereof, with or without a secondagent, are useful for ameliorating or reducing the symptoms of, ortreating, or preventing, the following non-limiting listing of diseasesand disorders: migraine (with or without aura), hemiplegic migraines,cluster headaches, migrainous neuralgia, chronic headaches, tensionheadaches, general headaches, hot flush, photophobia, chronic paroxysmalhemicrania, secondary headaches due to an underlying structural problemin the head or neck, cranial neuralgia, sinus headaches (e.g., headacheassociated with sinusitis), allergy-induced headaches or migraines,pain, chronic pain, neuroinflammatory or inflammatory pain,post-operative incision pain, post-surgical pain, trauma-related pain,eye pain, tooth pain, complex regional pain syndrome, cancer pain (e.g.,primary or metastatic bone cancer pain), fracture pain, osteoporoticfracture pain, pain resulting from burn, gout joint pain, painassociated with sickle cell crises, pain associated withtemporomandibular disorders, cirrhosis, hepatitis, neurogenic pain,neuropathic pain, nociceptic pain, visceral pain, trigeminal neuralgia,post-herpetic neuralgia, phantom limb pain, fibromyalgia, menstrualpain, ovarialgia, reflex sympathetic dystrophy, osteoarthritis orrheumatoid arthritis pain, lower back pain, diabetic neuropathy,sciatica, dyspepsia, irritable bowel syndrome, inflammatory boweldisease, Crohn's disease, ileitis, ulcerative colitis, renal colic,dysmenorrhea, cystitis, interstitial cystitis, menstrual period, labor,menopause, pancreatitis, schizophrenia, depression, post-traumaticstress disorder, anxiety disorders, diabetes, autoimmune diabetes,endothelial dysfunction, ischemia, Raynaud's syndrome, coronary heartdisease (“CHD”), coronary artery disease (“CAD”), heart failure,peripheral arterial disease (“PAD”), pulmonary hypertension (“PH”),connective tissue disorders, stroke, Sjögren's syndrome, multiplesclerosis, bronchial hyperreactivity, asthma, bronchitis,bronchodilation, emphysema, chronic obstructive pulmonary disease(“COPD”), inflammatory dermatitis, adenocarcinoma in glandular tissue,blastoma in embryonic tissue of organs, carcinoma in epithelial tissue,leukemia in tissues that form blood cells, lymphoma in lymphatic tissue,myeloma in bone marrow, sarcoma in connective or supportive tissue,adrenal cancer, AIDS-related lymphoma, anemia, bladder cancer, bonecancer, brain cancer, breast cancer, carcinoid tumors, cervical cancer,chemotherapy, colon cancer, cytopenia, endometrial cancer, esophagealcancer, gastric cancer, head cancer, neck cancer, hepatobiliary cancer,kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, Hodgkin'sdisease, non-Hodgkin's, nervous system tumors, oral cancer, ovariancancer, pancreatic cancer, prostate cancer, rectal cancer, skin cancer,stomach cancer, testicular cancer, thyroid cancer, urethral cancer,cancer of bone marrow, multiple myeloma, tumors that metastasize to thebone, tumors infiltrating the nerve and hollow viscus, tumors nearneural structures. Further preferably the cancer pain comprises visceralpain, preferably visceral pain which arises from pancreatic cancerand/or metastases in the abdomen. Further preferably the cancer paincomprises somatic pain, preferably somatic pain due to one or more ofmetastasis in the bone, postsurgical pain, sarcomas cancer of theconnective tissue, cancer of bone tissue, cancer of blood-forming cellsof the bone marrow, multiple myeloma, leukemia, primary or secondarybone cancer, acne vulgaris, atopic dermatitis, urticaria, keloids,hypertrophic scars and rosacea, allergic dermatitis, psoriasis,pruritus, neurogenic cutaneous redness, erythema, weight loss, anorexia,sarcoidosis, shock, sepsis, opiate withdrawal syndrome, morphinetolerance, epilepsy, lower urinary tract (“LUT”) disorders such asurinary tract infection, abnormal voiding, urinary urgency, nocturia,urinary incontinence, overactive bladder and for preventing oralleviating the pain associated with such LUT conditions. Preferably,the subject anti-PACAP antibodies and antigen binding fragmentsdescribed herein are useful for ameliorating or reducing the symptomsof, treating, or preventing migraine, headache and a pain associateddisease or condition.

In particular, the subject anti-PACAP antibodies and antigen bindingfragments can also be useful for ameliorating or reducing the symptomsof, treating, or preventing photophobia, occurring with a headacheand/or migraine as well as occurring independent of a headache and/or amigraine.

Migraineurs typically develop worsening pain and migraine symptoms whenexposed to light, a phenomenon known as photophobia. Photophobia is alsocommon in ocular disorders, such as iritis and uveitis, and intracranialdisorders, such as meningitis. In the classic visual pathway, lightactivates rods and cones in the retina, which activate retinal ganglioncells that project via the optic nerve, to the lateral geniculatenucleus, superior colliculus, and then the visual cortex. This pathwayincludes image-forming and non-image-forming data. A new pathway(non-image-forming information) allows maintenance of normal circadianrhythms via the suprachiasmatic nucleus and is regulated byintrinsically photosensitive retinal ganglion cells (ipRGCs). TheseipRGCs are independent of the rods and cones and contain melanopsin, aphotopigment.

Noseda, R. et al., Nat. Neurosci., 13:239-245 (2010) studied blindindividuals who had migraine and correlated these findings with ratmodels involving tracing of ipRGC projections to areas in perception ofpain from the dura. Of the blind patients with migraine, 6 had no lightperception due to severe optic nerve damage or bilateral enucleation.These subjects experienced abnormal sleep patterns and poor pupillarylight responses. Their migraines did not worsen with light exposure. Incontrast, 14 blind subjects who were able to detect light despiteminimal perception of images had normal sleep patterns and a normalpupillary light reflex. Despite widespread rod and cone degeneration,these patients had worsening migraine symptoms with light exposureduring migraine attacks, suggesting that ipRGCs, and not rods and cones,are important in photophobia.

These retinal projections of non-image-forming brain areas project tothe contralateral dorsocaudal region of the posterior thalamus, asdemonstrated by anterograde tracing in the rat. ipRGC input to this areamodulates dura-sensitive pain neurons, which also project to thisregion. Thalamic neurons, dually sensitive to dural pain and lightinput, project widely to multiple cortical regions, including theprimary somatosensory cortex, the primary and secondary motor cortices,the parietal association cortex, and the primary and secondary visualcortices. These cortical projections may help explain other commonmigraine symptoms, in addition to photophobia, such as motor weakness orincoordination, visual disturbances, and poor concentration.

Photophobia also accompanies other less frequent but likewise disablingconditions, such as cluster headache and other trigeminal autonomiccephalalgias and blepharospasm. The mechanisms underlying photophobiainvolve the trigeminal system. Photophobia in blind patients suggestscontributions from a nonvisual pathway. In addition, trigeminalautonomic cephalalgias, a less common group of primary headachedisorders, are characterized by unilateral trigeminal-mediated painfrequently associated with ipsilateral photophobia.

Common causes of photophobia include migraine headaches, cataracts, orsevere ophthalmologic diseases such as uveitis or corneal abrasion. Amore extensive list of disorders associated with photophobia includeseye related causes such as achromatopsia, aniridia, anticholinergicdrugs may cause photophobia by paralyzing the iris sphincter muscle,aphakia (absence of the lens of the eye), buphthalmos (abnormally narrowangle between the cornea and iris), cataracts, cone dystrophy,congenital abnormalities of the eye, viral conjunctivitis (“pink eye”),corneal abrasion, corneal dystrophy, corneal ulcer, disruption of thecorneal epithelium, such as that caused by a corneal foreign body orkeratitis, ectopia lentis, endophthalmitis, eye trauma caused bydisease, injury, or infection such as chalazion, episcleritis, glaucoma,keratoconus, or optic nerve hypoplasia, hydrophthalmos, or congenitalglaucoma iritis, optic neuritis, pigment dispersion syndrome, pupillarydilation (naturally or chemically induced), retinal detachment, scarringof the cornea or sclera and uveitis.

In addition, photophobia has nervous-system-related or neurologicalcauses including: autism spectrum disorders, Chiari malformation,dyslexia, encephalitis including myalgic encephalomyelitis aka chronicfatigue syndrome, meningitis, subarachnoid hemorrhage, tumor of theposterior cranial fossa, as well as other causes such as ankylosingspondylitis, albinism, ariboflavinosis, benzodiazepines (long term useof or withdrawal from benzodiazepines), chemotherapy, chikungunya,cystinosis, Ehlers-Danlos syndrome, hangover, influenza, infectiousmononucleosis, magnesium deficiency, mercury poisoning, migraine,rabies, and tyrosinemia type II, also known as “Richner-Hanhartsyndrome”.

Additionally, it is known that photophobia is elevated in depression,bipolar disorder and agoraphobia.

The subject anti-PACAP antibodies and antigen binding fragmentsdescribed herein can be effective for treating or preventing photophobiain any of these conditions, preferably, in a subject with post-traumaticstress disorder (“PTSD”) or in a subject with traumatic brain injury.

Headaches may be classified by cause, as discussed below.

Primary Headaches.

A primary headache is caused by problems with or overactivity ofpain-sensitive structures in the head. A primary headache is generallynot considered to be a symptom of an underlying disease. Instead,chemical activity in the brain, the nerves or blood vessels of the headoutside the skull, or muscles of the head and neck, or some combinationof these factors, may play a role in primary headaches. Some people maycarry genes that make them more likely to develop such headaches.Exemplary common primary headaches include, but are not limited to,cluster headache; tension headache (or tension-type headache); andtrigeminal autonomic cephalalgia (“TAC”), including paroxysmalhemicrania. There are other headache patterns that may be consideredtypes of primary headache, e.g., chronic daily headaches, coughheadaches, exercise headaches, and sex headaches. These headaches areless common and have distinct features, such as an unusual duration orpain associated with a certain activity. Although these headaches aregenerally considered primary, each of them could be a symptom of anunderlying disease. Additionally, some primary headaches can betriggered by lifestyle factors, including: alcohol; certain foods (e.g.,processed meats that contain nitrates); changes in sleep or lack ofsleep; poor posture; skipped meals; and stress.

Secondary Headaches.

A secondary headache is a symptom of a disease that can activate thepain-sensitive nerves of the head. Any number of conditions, which canvary greatly in severity, may cause secondary headaches. Exemplarysources of secondary headaches include, but are not limited to, acutesinusitis; arterial tears (carotid or vertebral dissections); venousthrombosis in the brain; brain aneurysm; brain arteriovenousmalformation; carbon monoxide poisoning; Chiari malformation;concussion; dehydration; dental problems; ear infection (middle ear);encephalitis; giant cell arteritis; glaucoma; hangovers; influenza(flu); intracranial hematoma; medications to treat other disorders;meningitis; monosodium glutamate (“MSG”); overuse of pain medication;panic attacks; post-concussion syndrome; pressure from tight-fittingheadgear, e.g., helmet or goggles; pseudotumor cerebri; toxoplasmosis;and trigeminal neuralgia. Specific types of secondary headaches include,but are not limited to, external compression headaches (a result ofpressure-causing headgear); ice cream headaches (commonly called “brainfreeze”); rebound headaches (caused by overuse of pain medication);sinus headaches (caused by inflammation and congestion in sinuscavities); spinal headaches (caused by low levels of cerebrospinalfluid, possibly the result of trauma, spinal tap or spinal anesthesia);and thunderclap headaches (a group of disorders that involves sudden,severe headaches).

Exemplary, non-limiting pain associated diseases and disorders that canbe treated and/or prevented by the administration of the anti-PACAPantibodies of the present invention include, pain resulting from anycondition associated with neurogenic, neuropathic, inflammatory, ornociceptic pain. Preferably, the pain-associated disorder will beassociated with increased PACAP at the pain site.

In certain embodiments, the pain associated disorder to be treated iscancer pain arising from malignancy or from cancer selected from one ormore of: adenocarcinoma in glandular tissue, blastoma in embryonictissue of organs, carcinoma in epithelial tissue, leukemia in tissuesthat form blood cells, lymphoma in lymphatic tissue, myeloma in bonemarrow, sarcoma in connective or supportive tissue, adrenal cancer,AIDS-related lymphoma, anemia, bladder cancer, bone cancer, braincancer, breast cancer, carcinoid tumors, cervical cancer, chemotherapy,colon cancer, cytopenia, endometrial cancer, esophageal cancer, gastriccancer, head cancer, neck cancer, hepatobiliary cancer, kidney cancer,leukemia, liver cancer, lung cancer, lymphoma, Hodgkin's disease,non-Hodgkin's, nervous system tumors, oral cancer, ovarian cancer,pancreatic cancer, prostate cancer, rectal cancer, skin cancer, stomachcancer, testicular cancer, thyroid cancer, urethral cancer, cancer ofbone marrow, multiple myeloma, tumors that metastasize to the bone,tumors infiltrating the nerve and hollow viscus, tumors near neuralstructures. Further preferably the cancer pain comprises visceral pain,preferably visceral pain which arises from pancreatic cancer and/ormetastases in the abdomen. Further preferably the cancer pain comprisessomatic pain, preferably somatic pain due to one or more of metastasisin the bone, postsurgical pain, sarcomas cancer of the connectivetissue, cancer of bone tissue, cancer of blood-forming cells of the bonemarrow, multiple myeloma, leukemia, primary or secondary bone cancer.

In other embodiments, the pain associated condition to be treated isassociated with neuropathic pain and included, by way of example,trigeminal neuralgia, post-herpetic neuralgia, phantom limb pain,fibromyalgia, and reflex sympathetic dystrophy are preferably treated.

Further exemplary pain associated diseases or conditions, include butare not limited to, general pain, chronic pain, inflammatory pain,post-operative incision pain, post-surgical pain, trauma-related pain,lower back pain, eye pain, tooth pain, complex regional pain syndrome,cancer pain (e.g., primary or metastatic bone cancer pain), fracturepain, osteoporotic fracture pain, pain resulting from burn, gout jointpain, pain associated with sickle cell crises, pain associated withtemporomandibular disorders, cirrhosis, hepatitis, neurogenic pain,neuropathic pain, nociceptic pain, visceral pain, trigeminal neuralgia,post-herpetic neuralgia, phantom limb pain, fibromyalgia, menstrualpain, ovarialgia, reflex sympathetic dystrophy, osteoarthritis orrheumatoid arthritis pain, lower back pain, diabetic neuropathy,sciatica, dyspepsia, irritable bowel syndrome, inflammatory boweldisease, Crohn's disease, ileitis, ulcerative colitis, renal colic,dysmenorrhea, cystitis, interstitial cystitis, menstrual period, labor,menopause, pancreatitis, schizophrenia, depression, post-traumaticstress disorder, anxiety disorders, diabetes, autoimmune diabetes,endothelial dysfunction, ischemia, Raynaud's syndrome, coronary heartdisease (“CHD”), coronary artery disease (“CAD”), heart failure,peripheral arterial disease (“PAD”), pulmonary hypertension (“PH”),connective tissue disorders, stroke, Sjögren's syndrome, multiplesclerosis, overactive bladder, bronchial hyperreactivity, asthma,bronchitis, bronchodilation, emphysema, chronic obstructive pulmonarydisease (“COPD”), inflammatory dermatitis, acne vulgaris, atopicdermatitis, urticaria, keloids, hypertrophic scars and rosacea, allergicdermatitis, psoriasis, puritus, neurogenic cutaneous redness, erythema,sarcoidosis, shock, sepsis, and opiate withdrawal syndrome.

Thus, the present invention includes methods of treating, preventing,and/or ameliorating any disease or disorder associated with PACAPactivity or PACAP upregulation (including any of the above mentionedexemplary pain associated diseases, disorders and conditions) throughuse of the antibodies and antigen binding fragments of the invention.

Also, the subject PACAP antibodies and antigen binding fragments may beused alone or in conjunction with other active agents, e.g., opioids andnon-opioid analgesics such as NSAIDs to elicit analgesia or topotentiate the efficacy of another analgesic.

The subject antibodies potentially may be combined with any opioidanalgesic or NSAID or other analgesic, potentially another antibody oranother biologic such as, e.g., an anti-NGF or anti-CGRP or anti-CGRP-Rantibody or antibody fragment or NGF, CGRP or CGRP-R polypeptidefragment or conjugate, in order to increase or enhance pain management.This may allow for such analgesic compounds to be administered forlonger duration or at reduced dosages thereby potentially alleviatingadverse side effects associated therewith.

Of particular interest is the co-administration of the subjectanti-PACAP antibodies and antibody fragments with an anti-CGRP antibody(e.g., ALD403) or or anti-CGRP-R antibody or antibody fragment and,moreover, the use of the subject anti-PACAP antibodies and antibodyfragments to treat subjects that previously received an anti-CGRP oranti-CGRP-R antibody or antibody fragment. For example, the previouslytreated subject (who previously received at least one anti-CGRP oranti-CGRP-R antibody or antibody fragment administration) may be amigraineur who did not adequately respond to anti-CGRP or anti-CGRP-Rantibody treatment (“poor responder”) and/or has elicited an immuneresponse to the anti-CGRP or anti-CGRP-R antibody or antibody fragment.

Likewise, the co-administration of the subject anti-PACAP antibodies andantigen binding fragments with BOTOX® (Botulinum toxin) is also ofparticular interest, e.g., in treating a migraineur. In some instances,the migraineur may not have adequately responded to previous treatments(“poor responder”) and/or has elicited an immune response to theprevious treatment.

In some embodiments, aspirin and/or acetaminophen may be taken inconjunction with the subject anti-PACAP antibody or antigen bindingfragment. Aspirin is another type of non-steroidal anti-inflammatorycompound.

The subject to which the pharmaceutical formulation is administered canbe, e.g., any human or non-human animal that is in need of suchtreatment, prevention and/or amelioration, or who would otherwisebenefit from the inhibition or attenuation of PACAP-mediated activity.For example, the subject can be an individual that is diagnosed with, orwho is deemed to be at risk of being afflicted by any of theaforementioned diseases or disorders. The present invention furtherincludes the use of any of the pharmaceutical formulations disclosedherein in the manufacture of a medicament for the treatment, preventionand/or amelioration of any disease or disorder associated with PACAPactivity (including any of the above mentioned exemplary diseases,disorders and conditions).

Administration

In one embodiment of the invention, the anti-PACAP antibodies describedherein, or PACAP binding fragments thereof, as well as combinations ofsaid antibodies or antigen binding fragments thereof, are administeredto a subject at a concentration of between 0.1 mg/ml and about any oneof 0.5, 1, 5, 10, 15 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200mg/ml, +/−10% error.

In another embodiment of the invention, the anti-PACAP antibodies andfragments thereof described herein are administered to a subject at adose of between about 0.01 and 100.0 or 200.0 mg/kg of body weight ofthe recipient subject. In certain embodiments, depending on the type andseverity of the PACAP-related disease, about 1 μg/kg to 50 mg/kg (e.g.,0.1-20 mg/kg) of antibody is an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. In anotherembodiment, about 1 μg/kg to 15 mg/kg (e.g., 0.1 mg/kg-10 mg/kg) ofantibody is an initial candidate dosage for administration to thepatient. A typical daily dosage might range from about 1 μg/kg to 100mg/kg or more, depending on several factors, e.g., the particular mammalbeing treated, the clinical condition of the individual patient, thecause of the disorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners. However, other dosage regimens may beuseful.

For example, in addition to the relative dosages (mg/kg) discussedherein, the subject anti-PACAP antibodies and antigen binding fragmentsthereof can be administered to a subject at an absolute dose (mg).Accordingly, in one embodiment of the invention, the anti-PACAPantibodies and antigen binding fragments thereof described herein areadministered to a subject at a dose of between about 1 microgram andabout 1000 milligrams regardless of the route of administration.

In a preferred embodiment of the invention, the anti-PACAP antibodiesdescribed herein, or anti-PACAP antigen binding fragments thereof, aswell as combinations of said antibodies or antigen binding fragmentsthereof, are administered to a recipient subject with a frequency ofonce every twenty-six weeks or less, such as once every sixteen weeks orless, once every eight weeks or less, once every four weeks or less,once every two weeks or less, once every week or less, or once daily orless.

According to preferred embodiments, the antibody containing medicamentor pharmaceutical composition is peripherally administered to a subjectvia a route selected from one or more of: orally, sublingually,buccally, topically, rectally, via inhalation, transdermally,subcutaneously, intravenously, intra-arterially, or intramuscularly, viaintracardiac administration, intraosseously, intradermally,intraperitoneally, transmucosally, vaginally, intravitreally,epicutaneously, intra-articularly, peri-articularly, or locally.

Fab fragments may be administered every two weeks or less, every week orless, once daily or less, multiple times per day, and/or every fewhours. In one embodiment of the invention, a patient receives Fabfragments of 0.1 mg/kg to 40 mg/kg per day given in divided doses of 1to 6 times a day, or in a continuous perfusion form, effective to obtaindesired results.

It is to be understood that the concentration of the antibody or Fabadministered to a given patient may be greater or lower than theexemplary administration concentrations set forth above.

A person of skill in the art would be able to determine an effectivedosage and frequency of administration through routine experimentation,for example guided by the disclosure herein and the teachings in,Goodman & Gilman's The Pharmacological Basis of Therapeutics, Brunton,L. L. et al. editors, 11^(th) edition, New York, New York: McGraw-Hill(2006); Howland, R. D. et al., Pharmacology, Volume 864, Lippincott'sillustrated reviews., Philadelphia, Pa.: Lippincott Williams & Wilkins(2006); and Golan, D. E., Principles of pharmacology: thepathophysiologic basis of drug therapy, Philadelphia, Pa.: LippincottWilliams & Wilkins (2007).

In another embodiment of the invention, the anti-PACAP antibodiesdescribed herein, or PACAP binding fragments thereof, as well ascombinations of said antibodies or antigen binding fragments thereof,are administered to a subject in a pharmaceutical formulation. In apreferred embodiment, the subject is a human.

A “pharmaceutical composition” or “medicament” refers to a chemical orbiological composition suitable for administration to a subject,preferably a mammal, more preferably a human. Such compositions may bespecifically formulated for administration via one or more of a numberof routes, including but not limited to buccal, epicutaneous, epidural,inhalation, intraarterial, intracardial, intracerebroventricular,intradermal, intramuscular, intranasal, intraocular, intraperitoneal,intraspinal, intrathecal, intravenous, oral, parenteral, rectally via anenema or suppository, subcutaneous, subdermal, sublingual, transdermal,and transmucosal. In addition, administration can occur by means ofinjection, powder, liquid, gel, drops, or other means of administration.

In one embodiment of the invention, the anti-PACAP antibodies describedherein, or PACAP binding fragments thereof, as well as combinations ofsaid antibodies or antigen binding fragments thereof, may be optionallyadministered in combination with one or more active agents. Such activeagents include analgesic, anti-histamine, antipyretic,anti-inflammatory, antibiotic, antiviral, and anti-cytokine agents.Active agents include agonists, antagonists, and modulators of TNF-α,IL-2, IL-4, IL-6, IL-10, IL-12, IL-13, IL-18, IFN-α, IFN-γ, BAFF,CXCL13, IP-10, VEGF, EPO, EGF, HRG, Hepatocyte Growth Factor (“HGF”),Hepcidin, NGF, CGRP including antibodies reactive against any of theforegoing, and antibodies reactive against any of their receptors.Active agents also include but are not limited to 2-arylpropionic acids,aceclofenac, acemetacin, acetylsalicylic acid (aspirin), alclofenac,alminoprofen, amoxiprin, ampyrone, arylalkanoic acids, azapropazone,benorylate/benorilate, benoxaprofen, bromfenac, carprofen, celecoxib,choline magnesium salicylate, clofezone, COX-2 inhibitors, dexibuprofen,dexketoprofen, diclofenac, diflunisal, droxicam, ethenzamide, etodolac,etoricoxib, faislamine, fenamic acids, fenbufen, fenoprofen, flufenamicacid, flunoxaprofen, flurbiprofen, ibuprofen, ibuproxam, indomethacin,indoprofen, kebuzone, ketoprofen, ketorolac, lornoxicam, loxoprofen,lumiracoxib, magnesium salicylate, meclofenamic acid, mefenamic acid,meloxicam, metamizole, methyl salicylate, mofebutazone, nabumetone,naproxen, N-arylanthranilic acids, NGF, oxametacin, oxaprozin, oxicams,oxyphenbutazone, oxytocin, parecoxib, phenazone, phenylbutazone,phenylbutazone, piroxicam, pirprofen, profens, proglumetacin,pyrazolidine derivatives, rofecoxib, salicyl salicylate, salicylamide,salicylates, substance P, sulfinpyrazone, sulindac, suprofen, tenoxicam,tiaprofenic acid, tolfenamic acid, tolmetin, and valdecoxib. Forinstance, the selected anti-PACAP antibodies, or PACAP-binding fragmentsthereof, as well as combinations of these antibodies or antigen bindingfragments, can be optionally administered in combination with oxytocin,for instance administered in a nasal formulation, for intranasaldelivery.

An anti-histamine can be any compound that opposes the action ofhistamine or its release from cells (e.g., mast cells). Anti-histaminesinclude but are not limited to acrivastine, astemizole, azatadine,azelastine, betatastine, brompheniramine, buclizine, cetirizine,cetirizine analogues, chlorpheniramine, clemastine, CS 560,cyproheptadine, desloratadine, dexchlorpheniramine, ebastine,epinastine, fexofenadine, HSR 609, hydroxyzine, levocabastine,loratadine, methscopolamine, mizolastine, norastemizole, phenindamine,promethazine, pyrilamine, terfenadine, and tranilast.

Antibiotics include but are not limited to amikacin, aminoglycosides,amoxicillin, ampicillin, ansamycins, arsphenamine, azithromycin,azlocillin, aztreonam, bacitracin, carbacephem, carbapenems,carbenicillin, cefaclor, cefadroxil, cefalexin, cefalothin, cefalotin,cefamandole, cefazolin, cefdinir, cefditoren, cefepime, cefixime,cefoperazone, cefotaxime, cefoxitin, cefpodoxime, cefprozil,ceftazidime, ceftibuten, ceftizoxime, ceftobiprole, ceftriaxone,cefuroxime, cephalosporins, chloramphenicol, cilastatin, ciprofloxacin,clarithromycin, clindamycin, cloxacillin, colistin, co-trimoxazole,dalfopristin, demeclocycline, dicloxacillin, dirithromycin, doripenem,doxycycline, enoxacin, ertapenem, erythromycin, ethambutol,flucloxacillin, fosfomycin, furazolidone, fusidic acid, gatifloxacin,geldanamycin, gentamicin, glycopeptides, herbimycin, imipenem,isoniazid, kanamycin, levofloxacin, lincomycin, linezolid, lomefloxacin,loracarbef, macrolides, mafenide, meropenem, methicillin, metronidazole,mezlocillin, minocycline, monobactams, moxifloxacin, mupirocin,nafcillin, neomycin, netilmicin, nitrofurantoin, norfloxacin, ofloxacin,oxacillin, oxytetracycline, paromomycin, penicillin, penicillins,piperacillin, platensimycin, polymyxin B, polypeptides, prontosil,pyrazinamide, quinolones, quinupristin, rifampicin, rifampin,roxithromycin, spectinomycin, streptomycin, sulfacetamide,sulfamethizole, sulfanilamide, sulfasalazine, sulfisoxazole,sulfonamides, teicoplanin, telithromycin, tetracycline, tetracyclines,ticarcillin, tinidazole, tobramycin, trimethoprim,trimethoprim-sulfamethoxazole, troleandomycin, trovafloxacin, andvancomycin.

Active agents also include aldosterone, beclomethasone, betamethasone,corticosteroids, cortisol, cortisone acetate, deoxycorticosteroneacetate, dexamethasone, fludrocortisone acetate, glucocorticoids,hydrocortisone, methylprednisolone, prednisolone, prednisone, steroids,and triamcinolone. Any suitable combination of these active agents isalso contemplated.

A “pharmaceutical excipient” or a “pharmaceutically acceptableexcipient” is a carrier, usually a liquid, in which an activetherapeutic agent is formulated. In one embodiment of the invention, theactive therapeutic agent is a humanized antibody described herein, orone or more fragments thereof. The excipient generally does not provideany pharmacological activity to the formulation, though it may providechemical and/or biological stability, and release characteristics.Exemplary formulations can be found, for example, in Remington'sPharmaceutical Sciences, Gennaro, A. editor, 19^(th) edition,Philadelphia, Pa.: Williams and Wilkins (1995), which is incorporated byreference.

As used herein “pharmaceutically acceptable carrier” or “excipient”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic, and absorption delaying agents that arephysiologically compatible. In one embodiment, the carrier is suitablefor parenteral administration. Alternatively, the carrier can besuitable for intravenous, intraperitoneal, intramuscular, or sublingualadministration. Pharmaceutically acceptable carriers include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. The use of such media and agents for pharmaceuticallyactive substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with the active compound,use thereof in the pharmaceutical compositions of the invention iscontemplated. Supplementary active compounds can also be incorporatedinto the compositions.

Pharmaceutical compositions typically must be sterile and stable underthe conditions of manufacture and storage. The invention contemplatesthat the pharmaceutical composition is present in lyophilized form. Thecomposition can be formulated as a solution, microemulsion, liposome, orother ordered structure suitable to high drug concentration. The carriercan be a solvent or dispersion medium containing, for example, water,ethanol, polyol (for example, glycerol, propylene glycol, and liquidpolyethylene glycol), and suitable mixtures thereof. The inventionfurther contemplates the inclusion of a stabilizer in the pharmaceuticalcomposition. The proper fluidity can be maintained, for example, by themaintenance of the required particle size in the case of dispersion andby the use of surfactants.

In many cases, it will be preferable to include isotonic agents, forexample, sugars, polyalcohols such as mannitol and sorbitol, or sodiumchloride in the composition. Absorption of the injectable compositionscan be prolonged by including an agent that delays absorption, forexample, monostearate salts and gelatin. Moreover, the alkalinepolypeptide can be formulated in a time-release formulation, for examplein a composition that includes a slow release polymer. The activecompounds can be prepared with carriers that will protect the compoundagainst rapid release, such as a controlled release formulation,including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, polylactic acid, polylactic and polyglycolic copolymers(“PLG”). Many methods for the preparation of such formulations are knownto those skilled in the art.

For each of the recited embodiments, the compounds can be administeredby a variety of dosage forms. Any biologically acceptable dosage formknown to persons of ordinary skill in the art, and combinations thereof,are contemplated. Examples of such dosage forms include, withoutlimitation, reconstitutable powders, elixirs, liquids, solutions,suspensions, emulsions, powders, granules, particles, microparticles,dispersible granules, cachets, inhalants, aerosol inhalants, patches,particle inhalants, implants, depot implants, injectables (includingsubcutaneous, intramuscular, intravenous, and intradermal), infusions,and combinations thereof.

The above description of various illustrated embodiments of theinvention is not intended to be exhaustive or to limit the invention tothe precise form disclosed. While specific embodiments of, and examplesfor, the invention are described herein for illustrative purposes,various equivalent modifications are possible within the scope of theinvention, as those skilled in the relevant art will recognize. Theteachings provided herein of the invention can be applied to otherpurposes, other than the examples described above.

These and other changes can be made to the invention in light of theabove detailed description. In general, in the following claims, theterms used should not be construed to limit the invention to thespecific embodiments disclosed in the specification and the claims.Accordingly, the invention is not limited by the disclosure, but insteadthe scope of the invention is to be determined entirely by the followingclaims.

The invention may be practiced in ways other than those particularlydescribed in the foregoing description and examples. Numerousmodifications and variations of the invention are possible in light ofthe above teachings and, therefore, are within the scope of the appendedclaims.

Certain teachings related to methods for obtaining a clonal populationof antigen-specific B-cells were disclosed in U.S. Patent PublicationNo. 2013/0316353, the disclosure of which is herein incorporated byreference in its entirety.

Certain teachings related to humanization of rabbit-derived monoclonalantibodies and preferred sequence modifications to maintainantigen-binding affinity were disclosed in International Publication No.WO 2008/144757, entitled Novel Rabbit Antibody Humanization Methods andHumanized Rabbit Antibodies, filed May 21, 2008, the disclosure of whichis herein incorporated by reference in its entirety.

Certain teachings related to producing antibodies or fragments thereofusing mating competent yeast and corresponding methods were disclosed inU.S. Patent Publication No. US2006/0270045, the disclosure of which isherein incorporated by reference in its entirety.

Certain teachings related to producing antibodies or fragments thereofin Pichia and preferred methods for obtaining and purifying antibodiesare also disclosed in U.S. Patent Publication Nos. 2014/0288272;2014/0287952; 2013/0055888; and 2012/0277408, the disclosures of each ofwhich are herein incorporated by reference in their entirety.

Certain teachings related to producing antibodies or fragments thereofin CHO cells and exemplary methods for obtaining and purifyingantibodies are also disclosed in U.S. Patent and Publication Nos. U.S.Pat. No. 7,932,087; 2009/0285795; U.S. Pat. No. 9,090,672; and2010/0221781; the disclosures of each of which are herein incorporatedby reference in their entirety.

Certain anti-PACAP antibody polynucleotides and polypeptides aredisclosed in the sequence listing accompanying this patent applicationfiling, and the disclosure of said sequence listing is hereinincorporated by reference in its entirety.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, manuals, books, or otherdisclosures) in the Background of the Invention, Detailed Description,and Examples is herein incorporated by reference in their entireties.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the subject invention, and are not intended to limit thescope of what is regarded as the invention. Efforts have been made toensure accuracy with respect to the numbers used (e.g. amounts,temperature, concentrations, etc.), but some experimental errors anddeviations should be allowed for. Unless otherwise indicated, parts areparts by weight, molecular weight is average molecular weight,temperature is in degrees centigrade; and pressure is at or nearatmospheric.

EXAMPLES Example 1 Preparation of Antibodies that Selectively Bind PACAP

By using an antibody selection protocol substantially as describedherein, a panel of antibodies specific to PACAP38 and PACAP27, and apanel of antibodies specific to PACAP38 only, were produced.

Immunization Strategy

Rabbits were immunized with PACAP38 (American Peptide, Vista, Calif.)(SEQ ID NO: 1241). Peptides were prepared for immunization as follows. A0.15 ml volume of 10 mg/ml keyhole limpet hemocyanin (“KLH”) dissolvedin Dulbecco's phosphate buffered saline (“DPBS”) supplemented to 1M NaClwas combined with 1.0 ml of 1 mg/ml peptide (dissolved in deionizedwater). Then 1.0 ml of 40 mM carbodiimide was added prior to a 12-hourincubation at room temperature with gentle mixing. Excess carbodiimideand unconjugated peptide were removed by dialysis to DPBS prior tosterile filtration. Next unconjugated peptide equal to the initial massof KLH was added prior to preparation for injection into rabbits.Alternatively, equal masses of sterile KLH and peptide were mixedwithout carbodiimide chemistry.

Immunizations were performed by diluting 200 μg of antigen to 0.5 mlwith DPBS and mixing with an equal volume of complete Freund's adjuvantfor subcutaneous 1 ml injection at Day 1.

Boost injections of 100 μg were performed with incomplete Freund'sadjuvant at Days 21 and 42.

Antibody Selection Functional Titer Assessment

To identify antibodies that neutralize PACAP38 (SEQ ID NO: 1241) inducedsignaling via PAC1-R, polyclonal antibody solutions were first purifiedvia Protein A and dialyzed into a neutral buffer. Briefly, antibodysolutions were incubated with PACAP38 (SEQ ID NO: 1241) at 4× the finalconcentration (100 pM) for 1 hr. While the antibody/antigen complexeswere incubated, PAC1-R expressing PC-12 cells (Japanese Collection ofResearch Bioresources Cell Bank) were washed and re-suspended at 2×10⁶cells per ml in cell culture media. Cells (10 μl) and antigen/antibodycomplex (40 μl) were transferred to a homogenous time resolvedfluorescence (“HTRF”) plate and shaken at room temperature for 30 min.Following the incubation, 20 μl of (1:20 diluted) Eu³⁺ cryptate-labeledmAb anti-cAMP and 20 μl of (1:20 diluted) d2-labeled cAMP in lysisbuffer were added, and the plate was incubated for 1 hr while shaking.Following incubation, plates were read (excitation 330 nm, emission620/665 nm), and a ratio of 620:665 signal was determined.

Tissue Harvesting

Once acceptable titers were established, the rabbit(s) were sacrificed.Spleen, lymph nodes, and whole blood were harvested and processed asfollows:

Spleen and lymph nodes were processed into a single cell suspension bydisassociating the tissue and pushing through sterile wire mesh at 70 μm(Thermo Fisher Scientific, Waltham, Mass.) with a plunger of a 20 ccsyringe. Cells were collected in phosphate buffered saline (“PBS”).Cells were then washed twice by centrifugation. After the last wash,cell density was determined by trypan blue. Cells were centrifuged at1500 RPM for 10 minutes; the supernatant was then discarded. Cells wereresuspended in the appropriate volume of 10% dimethyl sulfoxide (“DMSO”,Sigma-Aldrich Co., St. Louis, Mo.) in fetal bovine serum (“FBS”HYCLONE™, GE Healthcare Life Sciences, Marlborough, Mass.) and dispensedat 1 ml/vial. Vials were stored at −70° C. in a slow freezing chamberfor 24 hours and stored in liquid nitrogen.

Peripheral blood mononuclear cells (“PBMCs”) were isolated by mixingwhole blood with equal parts of PBS. 35 ml of the whole blood mixturewas carefully layered onto 8 ml of LYMPHOLYTE® Rabbit (CedarlaneLaboratories, Burlington, Ontario) into a 45 ml conical tube (Corning,Corning, N.Y.) and centrifuged for 30 minutes at 2500 RPM at roomtemperature without brakes. After centrifugation, the PBMC layers werecarefully removed using a glass Pasteur pipette (VWR International,Radnor, Pa.), combined, and placed into a clean 50 ml vial. Cells werewashed twice with PBS by centrifugation at 1500 RPM for 10 minutes atroom temperature, and cell density was determined by trypan bluestaining. After the last wash, cells were resuspended in an appropriatevolume of 10% DMSO/FBS medium and frozen as described above.

B-Cell Selection, Enrichment, and Culture Conditions

On the day of setting up B-cell culture, PBMC, splenocyte, or lymph nodevials were thawed for use. Vials were removed from liquid nitrogen tankand placed in a 37° C. water bath until thawed. Contents of vials weretransferred into 15 ml conical centrifuge tube (Corning, Inc., Corning,N.Y.) and 10 ml of modified RPMI was slowly added to the tube. Cellswere centrifuged for 5 minutes at 2000 RPM, and the supernatant wasdiscarded. Cells were resuspended in 10 ml of fresh media. Cell densityand viability was determined by trypan blue.

For positive selection of anti-PACAP38 producing B-cells, biotinylatedPACAP38 (SEQ ID NO: 1241) was pre-loaded onto the streptavidin beads asfollows. 75 μl of streptavidin beads (Miltenyi Biotec, Auburn, Calif.)were mixed with N-terminally biotinylated PACAP38 (10 μg/ml finalconcentration) and 300 μl of PBS supplemented with 0.5% biotin freebovine serum albumin (“BSA”) and 2 mM EDTA (“PBF”). This mixture wasincubated at 4° C. for 30 minutes, and unbound biotinylated PACAP38(AnaSpec, Fremont, Calif.) was removed using a MACS® separation column(Miltenyi Biotec, Auburn, Calif.) with a 1 ml rinse to remove unboundmaterial. The bound material was plunged out by detachment from themagnet and used to resuspend cells from above in 100 μl per 1×10⁷ cells.The mixture was then incubated at 4° C. for 30 minutes and washed oncewith 10 ml of PBF. After washing, the cells were resuspended in 500 μlof PBF and set aside. A MACS® MS column (Miltenyi Biotec, Auburn,Calif.) was pre-rinsed with 500 μl of PBF on a magnetic stand (MiltenyiBiotec, Auburn, Calif.). Cell suspension was applied to the columnthrough a pre-filter, and unbound fraction was collected. The column waswashed with 2.5 ml of PBF buffer. The column was removed from the magnetstand and placed onto a clean, sterile 1.5 ml EPPENDORF™ tube. 1 ml ofPBF buffer was added to the top of the column, and positive selectedcells were collected. The yield and viability of positive cell fractionwas determined by trypan blue staining. Positive selection yielded anaverage of 1% of the starting cell concentration.

A pilot cell screen was established to provide information on seedinglevels for the culture. Plates were seeded at 5, 10, 25, 50, 100, or 200enriched B-cells/well. In addition, each well contained 25-50Kcells/well of irradiated EL-4.B5 cells (5,000 Rads) and an appropriatelevel of activated rabbit T-cell supernatant (See U.S. PatentApplication Publication No. 20070269868) (ranging from 1-5% depending onpreparation) in high glucose modified RPMI medium at a final volume of250 μl/well. Cultures were incubated for 5 to 7 days at 37° C. in 4%CO₂.

B-Cell Culture Screening by Antigen-Recognition (ELISA)

To identify wells producing anti-PACAP38 antibodies, B-cell supernatantswere tested by antigen-recognition (ELISA). Briefly, NEUTRAVIDIN™-coatedplates (Thermo Fisher Scientific, Waltham, Mass.), were coated witheither N-term or C-term biotinylated PACAP38 (AnaSpec Inc., Fremont,Calif.) (50 μl per well; 1 μg/ml) diluted in ELISA buffer (0.5% fishskin gelatin in PBS pH 7.4) either for approximately 1 hour at roomtemperature or alternatively overnight at 4° C. The plates were thenfurther blocked with ELISA buffer for one hour at room temperature andwashed using PBS with 0.05% Tween 20 (“wash buffer”). B-cell supernatantsamples (50 μl) were transferred onto the wells and incubated for onehour at room temperature. After this incubation, the plate was washedwith wash buffer. For development, an anti-rabbit specific Fc-HorseRadish Peroxidase (“Fc-HRP”) (1:5000 dilution in ELISA buffer) was addedonto the wells and incubated for 45 minutes at room temperature. After a3× wash step with wash solution, the plate was developed using3,3′,5,5′-Tetramethylbenzidine (“TMB”) substrate for two minutes at roomtemperature, and the reaction was quenched using 0.5M HCl. The wellabsorbance was read at 450 nm.

To identify wells producing anti-PACAP38 antibodies that do notrecognize VIP (SEQ ID NO: 1243), supernatant from wells positive forPACAP38 binding by ELISA were tested by ELISA for binding to VIP.Briefly, biotinylated VIP (AnaSpec Inc., Fremont, Calif.) was bound ontoNEUTRAVIDIN™ coated plates (50 μg per well, 1 μg/μl each peptide).B-cell supernatant samples (50 μl) were tested without prior dilution.Recognition in this assay may indicate cross reactivity with a closelyrelated peptide, VIP.

Identification of Functional Activity in B-Cell Supernatants Using Oneor More Assays

To identify wells producing anti-PACAP38 antibodies that block signalingof PACAP38 via PAC1-R, supernatant from positive wells for PACAP38binding by ELISA were tested in a cAMP HTRF assay (Cisbio US, Bedford,Mass.). Supernatants (78 μl) were pre-incubated with 2 μl 5 nM PACAP38(American Peptide Company, Sunnyvale, Calif.) for 1 hour at 37° C.During the incubation, PC-12 cells were prepared as described for titerassessment. Cells (10 μl) and antigen/antibody complex (40 μl) weretransferred to an HTRF plate and shaken at room temperature for 30minutes. Following the incubation, 20 μl of (1:20 diluted) Eu³⁺cryptate-labeled mAb anti-cAMP and 20 μl of (1:20 diluted) d2-labeledcAMP in lysis buffer were added, and the plate was incubated for 1 hourwhile shaking. Following incubation plates were read (excitation 330 nm,emission 620/665 nm), and a ratio of 620:665 signal was determined.

Isolation of Antigen-Specific B-cells

Antigen-specific B-cells were isolated (for general methods see co-ownedpublication no. WO 2014/146074, which is hereby incorporated byreference in its entirety). Plates containing wells of interest wereremoved from −70° C., and the cells from each well were recovered usingfive washes of 200 μl of medium (10% RPMI complete, 55 μMβ-mercaptoethanol (“BME”)) per well. The recovered cells were pelletedby centrifugation and the supernatant was carefully removed. Cells fromeach well were then re-suspended in 100 μl of medium and transferred toa 96 well plate. Cells were incubated for 90 minutes at 37° C. Followingincubation, cells were pelleted by centrifugation, stained with afluorescein isothiocyanate-labeled (“FITC-labeled”) anti-rabbit IgG(final concentration 6.25 μg/ml) (Creative Diagnostics, Shirley, N.Y.),and washed with up to 2 ml fluorescence-activated cell sorting buffer(“FACS buffer”) (Dulbecco's PBS w/2% FBS) and re-suspended in 250 μl ofFACS buffer.

Control wells from the same culture sets that were similar incomposition to pooled wells of interest were thawed and stainedalongside target wells. These samples were initially run on FACS (BDINFLUX™, Becton, Dickinson and Company, Franklin Lakes, N.J.), and gateswere established for IgG, viability, and physical parameters (Forwardscatter (“FSC”)/side scatter (“SSC”)) that differentiate B-cells fromthe murine EL4 cells. Once gates were established, the sample ofinterest was run, and IgG positive, viable cells that were of aconsistent physical (FSC/SSC) population were sorted individually intowells of a 96 well plate pre-loaded with RT-PCR master mix. Upwards of 8cells per well were sorted. Sorted plates were removed from the sorterand transferred directly to thermocyclers for PCR.

Amplification and Sequence Determination of Antibody Sequences FromFACS-Sorted B-Cells

Antibody sequences were recovered using a combined RT-PCR based methodfrom a single cell sorted B-cell. Primers containing restriction enzymeswere designed to anneal in conserved and constant regions of the targetimmunoglobulin genes (heavy and light), such as rabbit immunoglobulinsequences, and a two-step nested PCR recovery was used to amplify theantibody sequence. Amplicons from each well were sequenced and analyzed.Representative antibodies from the resulting sequence clusters wereselected for recombinant protein expression. The original heavy andlight variable regions amplified from rabbit cells were cloned intohuman heavy and light chain constant region expression vectors viarestriction enzyme digestion and ligation, and via Gibson method.Vectors containing subcloned DNA fragments were amplified and purified.The sequences of the subcloned heavy and light chains were verifiedprior to expression.

Recombinant Production of Monoclonal Antibody of Desired AntigenSpecificity and/or Functional Properties

To determine antigen specificity and functional properties of recoveredantibodies from specific B-cells, the heavy and light chain plasmidswere co-transfected to generate rabbit/human chimeric antibodies fortesting. Briefly, heavy and light chimeric plasmids were transientlytransfected into HEK-293 cells. Transfections were allowed to incubatefor 5-7 days, and upon harvest, cells were pelleted by centrifugation.Supernatants were submitted for purification via Protein A. Resultingpurified chimeric antibodies were then evaluated in a variety of assaysto confirm specificity and potency.

Using the above-described methods, numerous functional (antagonistic)antibodies that bind PACAP38 and PACAP27, or that bind PACAP38 only, butwhich do not, or do not appreciably, bind to VIP were identified.Polypeptide and exemplary coding sequences of exemplary antagonisticanti-PACAP antibodies are contained in the included biological sequencelisting.

The full-length antibodies Ab10, Ab20, Ab21, Ab22, and Ab23 used inthese examples were expressed as the heavy chain polypeptides having thesequences of SEQ ID NO: 401; 441; 841; 881; and 921, respectively, andthe light chain polypeptides of SEQ ID NO: 421; 461; 861; 901; and 941,respectively. The heavy chain polypeptides of antibodies Ab10, Ab20,Ab21, Ab22, and Ab23 were expressed from the polynucleotides of SEQ IDNO: 411; 451; 851; 891; and 931, respectively. The light chainpolypeptides of antibodies Ab10, Ab20, Ab21, Ab22, and Ab23 wereexpressed from the polynucleotides of SEQ ID NO: 431; 471; 871; 911; and951, respectively. Additional features of said antibodies are identifiedby SEQ ID NOS in FIGS. 1-12.

Antigen Binding Specificity of Antibodies by Competitive HTRF BindingAssay

The binding and functional properties of exemplary anti-PACAP38 andanti-PACAP27 antibodies produced according to the invention are furtherdescribed below.

To identify antibodies that preferentially bind PACAP38 (SEQ ID NO:1241) and PACAP27 (SEQ ID NO: 1242), but do not bind VIP (SEQ ID NO:1243), or to identify antibodies that specifically bind PACAP38, but donot bind appreciably PACAP27, or do not appreciably bind VIP, etc., acompetition HTRF binding assay was performed.

In parallel, 10 μl of an antibody dilution series (highest finalconcentration of 100 nM) were incubated with 10 μl of N-terminal orC-terminal biotinylated PACAP38 (35 nM final) alone, or in combinationwith either PACAP27 (350 nM final) or VIP (350 nM final), i.e., 10×PACAP27 or 10×VIP, respectively, in a HTRF plate. 20 μl of Eu³⁺ cryptatelabeled anti-hu Fc donor and 20 μl of d2-labeled streptavidin acceptorwere added to each well and incubated for 1 hour at room temperature.Fluorescence was measured at 620 and 665 nm with a delay of 300 μsec.

FIG. 13A-D provide representative binding data for Ab10, Ab20, Ab21, andAb1.H to PACAP38 and to PACAP27, and the inability of VIP to competewith binding of PACAP38. FIG. 13E and FIG. 13F provide representativebinding data for the anti-PACAP antibodies Ab22 and Ab23 to PACAP38 andthe inability of PACAP27 or VIP to compete with binding of PACAP38. Thelack of effect of VIP on binding to PACAP38 indicated its inability tocompete with binding of PACAP38. These results demonstrated that Ab10,Ab20, Ab21, and Ab1.H bind to PACAP38 and PACAP27, but do not bind (ordo not appreciably bind) VIP. These results also demonstrated that Ab22and Ab23 bind to PACAP38, but do not bind (or do not appreciably bind)PACAP27 or VIP.

EC₅₀ values, i.e. the concentration of an antibody that yields aresponse halfway between the baseline and the maximum value within aspecified time period, were computed for each antibody based upon theirbinding curves and are shown in Table 1 below. The results demonstratethat Ab10, Ab20, Ab21, Ab22, and Ab23 bound to and recognized humanPACAP38 with high affinity. A humanized form of antibody Ab1 identifiedby an appended “.H”, i.e., Ab1.H also bound PACAP38 with high affinity.

TABLE 1 Binding (EC₅₀) of PACAP38 by anti-PACAP antibodiesPACAP38-binding ANTIBODY EC50 (nM) Ab10 0.36 Ab20 0.38 Ab21 0.84 Ab1.H0.46 Ab22 0.57 Ab23 0.56Ability of anti-PACAP Antibodies to Neutralize PACAP38-Induced andPACAP27-Induced cAMP Production

The ability of anti-PACAP antibodies to neutralize PACAP38-induced andPACAP27-induced PAC1-R signaling was tested in a cell-based assay.

For Ab10, Ab20, Ab21, Ab1.H, Ab10.H, Ab21.H, Ab22, and Ab23 to identifyantibodies that neutralized PACAP38-induced and PACAP27-inducedsignaling via PAC1-R, antibody solutions were incubated with eitherPACAP38 or with PACAP27 at 4× the final concentration (100 pM) for 1hour. While the antibody/antigen complexes were incubated, PAC1-Rexpressing PC-12 cells (Japanese Collection of Research BioresourcesCell Bank) were washed and re-suspended at 2×10⁶ cells per ml in cellculture media. Cells (10 μl) and antigen/antibody complex (40 μl) weretransferred to an HTRF plate and shaken at room temperature for 30minutes. Following the incubation, 20 μl of (1:20 diluted) Eu³⁺cryptate-labeled mAb anti-cAMP and 20 μl of (1:20 diluted) d2-labeledcAMP in lysis buffer were added, and the plate was incubated for 1 hourwhile shaking. Following incubation, plates were read (excitation 330nm, emission 620/665 nm), and a ratio of 620:665 signal was determined.The final concentration of PACAP38 and PACAP27 in each well was 0.1 nM.

FIGS. 16A-H (PACAP38) and FIGS. 17A-H (PACAP27) show inhibition curves(for Ab10, Ab20, Ab21, Ab1.H, Ab10.H, Ab21.H, Ab22, and Ab23) that arerepresentative of the inhibition curves that were obtained with thetested antibodies. The inhibition results were quantified for eachantibody to yield an IC₅₀ value, which are summarized in Table 2 below.These results demonstrated that anti-PACAP antibodies Ab10, Ab20, Ab21,Ab1.H, Ab10.H, Ab21.H, Ab22, and Ab23 inhibited PACAP38-induced cAMPincrease in cells expressing PAC1-R (see FIGS. 16A-H). Additionally,these results demonstrated that anti-PACAP antibodies Ab10, Ab20, Ab21,Ab1.H, Ab10.H, and Ab21.H, but not Ab22 or Ab23, inhibitedPACAP27-induced cAMP increase in cells expressing PAC1-R (see FIGS.17A-H).

TABLE 2 Inhibition (IC₅₀) of PACAP38-induced and PACAP27-induced cAMPincrease in cells expressing PAC1-R by anti-PACAP antibodies Inhibitionof 0.1 nM Inhibition of 0.1 PACAP38-induced nM PACAP27- PAC1-R mediatedinduced PAC1-R cAMP increase IC₅₀ mediated cAMP ANTIBODY (pM) increaseIC₅₀ (pM) Ab10 180.3 227.0 Ab20 368.2 187.8 Ab21 239.1 140.2 Ab1.H 259.657.7 Ab10.H 163.4 84.0 Ab21.H 246.0 203.6 Ab22 101.4 n/a * Ab23 114.9n/a * * n/a: not active because these Abs are PACAP38 specific

Example 2 Binding Affinities of Anti-PA CAP Antibodies

Binding affinities of monoclonal antibodies for human PACAP wereestimated using SPR on the PROTEON™ XPR36 (Bio-Rad, Hercules, Calif.).Antibody was immobilized to the surface of general amine coupling (“GLC”or “GLM”) Chips (Bio-Rad, Hercules, Calif.). A dilution series of humanPACAP38 (SEQ ID NO: 1241) prepared in 1×PBST Buffer (4.3 mM NaPhosphate, 1.4 mM K Phosphate, 135 mM NaCl, 2.7 mM KCl 0.05%Polysorbate-20) purchased from Teknova (Cat# P1192, Teknova, Hollister,Calif.) and supplemented with 0.25 M arginine (from J. T. BAKER®), 0.2mg/ml BSA (Jackson Immuno Research Labs, West Grove, Pa.), and 0.005%sodium azide (VWR International, Radnor, Pa.) with the pH adjusted to 7was used to query the antibodies. Antigen (ranging from 1.23 nM to 100nM) was typically run sequentially with association times of 2-4 minutesand dissociation times of 3-120 minutes grouped with the PROTEON™Manager Software (v3.1.0.6 (Bio-Rad, Hercules, Calif.)) and fitted usinga 1:1 Langmuir binding model. Surfaces were regenerated between analytequeries using 0.85% Phosphoric Acid. A single K_(D) was calculated foreach antibody with association times limited near the rate of diffusion(1.0×10⁶) and dissociation times limited to 1.5×10⁻⁵ where nodiscernible dissociation was observed.

The same procedure was used to determine binding affinities ofantibodies for human VIP (SEQ ID NO: 1243) and PACAP27 (SEQ ID NO: 1242)though peptide concentrations ranged from 1.23 nM to 1000 nM withassociation times of 200 seconds and dissociation times of 3-120minutes.

The measured antibody affinities for PACAP38 are listed in Table 3.

TABLE 3 Antibody affinity constants for PACAP38 Antibody ka (1/Ms) kd(1/s) KD (M) Ab10 2.6E+05 2.0E−05 7.5E−11 Ab20 1.2E+05 2.4E−05 2.1E−10Ab21 3.7E+05 1.0E−05 2.7E−11 Ab22 3.7E+05 1.0E−05 2.7E−11 Ab23 5.1E+053.6E−05 7.1E−11 Ab1.H 4.7E+05 1.0E−05 2.1E−11 Ab10.H 3.4E+05 1.0E−052.9E−11 Ab21.H 4.8E+05 1.0E−05 2.1E−11

Examples of antibody affinity constants for VIP are listed in Table 4.

TABLE 4 Antibody affinity constants for VIP Antibody ka (1/Ms) kd (1/s)KD (M) Ab10 3.7E+04 1.0E−02 2.8E−07 Ab20 4.5E+05 4.8E−01 1.1E−06 Ab211.7E+03 5.4E−04 3.1E−07 Ab22 2.7E+05 1.8E−01 6.9E−07 Ab23 4.3E+053.2E−01 7.3E−07 Ab1.H 3.8E+04 1.8E−01 4.8E−06 Ab10.H 3.8E+05 3.9E−021.0E−07 Ab21.H 1.4E+05 5.9E−02 4.4E−07

Examples of antibody affinity constants for PACAP27 are listed in Table5.

TABLE 5 Antibody affinity constants for PACAP27 Antibody ka (1/Ms) kd(1/s) KD (M) Ab10 1.0E+06 1.0E−05 1.0E−11 Ab20 3.2E+05 2.2E−05 7.0E−11Ab21 7.9E+05 1.0E−05 1.3E−11 Ab22 1.0E+00 1.0E−01 1.0E−01 Ab23 8.9E+053.1E−02 3.5E−08 Ab1.H 7.6E+05 1.0E−05 1.3E−11 Ab10.H 5.3E+05 1.8E−053.3E−11 Ab21.H 6.3E+05 1.0E−05 1.6E−11

The binding affinity results of Tables 3 and 5 present datademonstrating that Ab23 weakly bound to PACAP27 as compared to itsbinding affinity for PACAP38. Tables 3 and 5 additionally present datademonstrating that Ab22 did not specifically recognize PACAP27, but thatAb22 specifically bound to PACAP38.

Example 3 Inhibition of PACAP38-Induced Signaling Via VPAC1-R

To identify antibodies that neutralize PACAP38-induced signaling viahuman VPAC1-R, CHO-K1 cells expressing human VPAC1-R were used in a cAMPHTRF cell-based assay. Antibody dilutions were incubated with PACAP38 at4× the final concentration (5 nM) for 1 hour. While the antibody/antigencomplexes were incubated for 1 hour, VPAC1-R expressing CHO-K1 cells(generated at Alder Biopharmaceuticals, by stable transfection of CHO-K1cells (ATCC, catalog # CCL-61) with human VPAC1-R cDNA; selected clone 1was used for in vitro cell based assays) were detached with 0.25%trypsin for 4 minutes. The cells were washed and re-suspended at 1×10⁶cells per ml culture media. 20 μl of Ab/antigen mixture was mixed with20 μl of cells in HTRF plates and incubated with shaking for 30 minutes.20 μl of Eu³⁺ cryptate labeled anti-cAMP mAb (1:20 diluted) and 20 μl of(1:20 diluted) d2-labeled cAMP in lysis buffer were added to each welland incubated for 1 hour with shaking. The final concentration ofPACAP38 in each well was 5 nM. Following incubation, plates were read(excitation 330 nm, emission 620/665 nm), and a ratio of 620:665 signalwas determined.

FIGS. 18A-H are representative of the inhibition curves obtained by thismethod (results are shown for Ab10, Ab20, Ab21, Ab1.H, Ab10.H, Ab21.H,Ab22, and Ab23 respectively). The computed IC₅₀ values for eachantibody, which are shown below in Table 6, demonstrated that Ab10,Ab20, Ab21, Ab1.H, Ab10.H, Ab21.H, Ab22, and Ab23 inhibitedPACAP38-induced cAMP increase in cells expressing human VPAC1-R.

TABLE 6 Inhibition (IC₅₀) of PACAP38-induced cAMP increase in cellsexpressing human VPAC1-R by anti-PACAP antibodies Inhibition of 5 nMPACAP38-induced human VPAC1-R mediated cAMP increase ANTIBODY IC50 (pM)Ab10 649.1 Ab20 3889.0 Ab21 2846.0 Ab1.H 1021.1 Ab10.H 1336.0 Ab21.H2105.0 Ab22 1300.0 Ab23 2667.0

Example 4 Inhibition of PACAP38-Induced Signaling Via VPAC2-R

To identify antibodies that neutralize PACAP38-induced signaling viahuman VPAC2-R, CHO-K1 cells expressing human VPAC2-R were used in a cAMPHTRF cell based assay. Antibody dilutions were incubated with PACAP38 at4× the final concentration (1 nM) for 1 hr. While the antibody/antigencomplexes were incubated for 1 hour, VPAC2-R expressing CHO-K1 cells(generated at Alder Biopharmaceuticals, by stable transfection of CHO-K1cells (ATCC, catalog # CCL-61) with human VPAC2-R cDNA; selected clone 8was used for in vitro cell based assays) were detached with 0.25%trypsin for 4 minutes. The cells were washed and re-suspended at 1×10⁶cells per ml culture media. 20 μl of Ab/antigen mixture was mixed with20 μl of cells in HTRF plates and incubated with shaking for 30 minutes.20 μl of Eu³⁺ cryptate labeled anti-cAMP mAb (1:20 diluted) and 20 μl of(1:20 diluted) d2-labeled cAMP in lysis buffer were added to each welland incubated for 1 hour with shaking. The final concentration ofPACAP38 in the wells was 1 nM. Following incubation, plates were read(excitation 330 nm, emission 620/665 nm) and, a ratio of 620:665 signalwas determined.

FIGS. 19A-H are representative of the inhibition curves obtained by thismethod (results are shown for Ab10, Ab20, Ab21, Ab1.H, Ab10.H, Ab21.H,Ab22, and Ab23 respectively). The computed IC₅₀ values for eachantibody, which are shown below in Table 7, demonstrated that Ab10,Ab20, Ab21, Ab1.H, Ab10.H, Ab21.H, Ab22, and Ab23 inhibitedPACAP38-induced cAMP increase in cells expressing human VPAC2-R.

TABLE 7 Inhibition (IC₅₀) of PACAP38-induced cAMP increase in cellsexpressing human VPAC2-R by anti-PACAP antibodies Inhibition of 1 nMPACAP38-induced human VPAC2-R mediated cAMP ANTIBODY increase IC₅₀ ( pM)Ab10 188.5 Ab20 14570.0 Ab21 5215.0 Ab1.H 983.0 Ab10.H 988.0 Ab21.H1507.0 Ab22 515.0 Ab23 1789.0

Example 5 Inhibition of PACAP38 Binding to PAC1-R-Expressing Cells

To identify antibodies that block PACAP38 binding to PAC1-R-expressingcells, adherent PC-12 cells (ATCC, Manassas, Va.) expressing PAC1-R wereused in a Europium-based PAC1-R-expressing cells binding assay. Antibodysolutions were incubated with N-terminal biotinylated PACAP38 at 10× thefinal concentration (100 nM or 30 nM) for 1 hr, then added to PC-12cells that were plated 24 hrs prior in black clear bottom 96 well plates(COSTAR™, Corning Incorporated, Corning, N.Y.) and further incubated for1 hr at room temperature. After three washes, the cells were incubatedwith 20 μL Europium-labeled streptavidin (PerkinElmer, Waltham, Mass.)for 1 hr at room temperature. Cells were washed three times, then 20 μlDELFIA® Enhancement solution (PerkinElmer, Waltham, Mass.) was added toeach well and incubated for 15 minutes with gentle shaking. Plates wereread (Time Resolved Fluorescence (“TRF”)) on SPECTRAIVIAX® (MolecularDevices, Sunnyvale, Calif.) plate reader.

FIGS. 14A-H are representative of the inhibition curves obtained by thismethod (results are shown for Ab10, Ab20, Ab21, Ab1.H, Ab10.H, Ab21.H,Ab22, and Ab23, respectively) wherein the PAC1-R expressing cells werePC-12 cells. The computed IC₅₀ values for each antibody, which are shownbelow in Table 8, demonstrated that Ab10, Ab20, Ab21, Ab1.H, Ab10.H,Ab21.H, Ab22, and Ab23 inhibited PACAP38 binding to PAC1-R expressingPC-12 cells.

TABLE 8 Inhibition (IC₅₀) of PACAP38 binding to PAC1-R-expressing PC-12cells by anti-PACAP antibodies ANTIBODY Inhibition of 100 nMbiotinylated PACAP38 binding to PAC1-R-expressing PC-12 cells IC50 (nM)Ab10 17.8 Ab20 32.7 Inhibition of 30 nM Biotinylated PACAP38 binding toPAC1-R-expressing PC-12 cells IC₅₀ (nM) Ab21 20.3 Ab1.H 56.3 Ab10.H 14.5Ab21.H 12.7 Ab22 13.8 Ab23 14.9

Example 6 PACAP38-Mediated Binding of Anti-PACAP Antibodies to the CellSurface of PAC1-R-Expressing Cells

To identify anti-PACAP antibodies that bind, via PACAP38, to the cellsurface of PAC1-R expressing cells, adherent PC-12 cells (JapaneseCollection of Research Bioresources Cell Bank) expressing PAC1-R wereused in a cell surface binding-based assay. To perform the bindingexperiment, PAC1-R expressing PC-12 cells were first seeded into Corning96 well white solid bottom plates (Corning, Corning, N.Y.). Cells wereinitially seeded at 1×10⁵ cells/well in a solution of complete RPMI(“cRPMI”: RPMI medium supplemented with 10% sterile heat-inactivated FBSand 1% sterile antibiotic/antimycotic)+10% FBS, and the plates wereallowed to incubate overnight at 37° C. On the day of the binding assay,antibodies at an initial concentration of 15 μg/ml were diluted at a 1:3ratio in DELFIA® binding buffer (50 mM Tris, 150 mM NaCl, 0.1% azide, 2%horse serum) (Perkin-Elmer, Waltham, Mass.) to a total volume of 60 μLin a separate 96 well round bottom plate. PACAP38 was prepared for thebinding assay by diluting it in DELFIA® binding buffer to aconcentration of 200 nM, and then 60 μl of the diluted PACAP38 was addedto each of the antibody-containing wells to form antibody:antigencomplexes. Following addition of PACAP38, the antibody:antigen complexeswere incubated at room temperature on a shaker for 1 hour. Separately,the PC-12 cells were prepared for addition of antibody:antigen complexesby washing the cells two times with DELFIA® wash buffer (50 mM Tris, 150mM NaCl, 0.1% Azide) (Perkin-Elmer, Waltham, Mass.). After washing thecells two times and following the 1 hour room temperature incubation ofthe antibody:antigen complexes, 50 μl of the antibody:antigen complexwas added to each well containing cells. The mixtures of cells andantibody:antigen complexes were then incubated for 30 minutes at roomtemperature. Following this 30 minute incubation, each mixture waswashed two times with DELFIA® wash buffer (Perkin-Elmer, Waltham,Mass.).

DELFIA® Europium labeled anti-human IgG detection reagent (Cat#1244-330, Perkin-Elmer, Waltham, Mass.) was diluted to a concentrationof 300 ng/ml in DELFIA® Binding Buffer. Following dilution, 50 μl of theanti-human IgG detection reagent was added to each well containingcells, and a 30 minute incubation at room temperature followed thisaddition of IgG detection reagent. After completion of the 30 minuteroom temperature incubation, the cells were then washed two times withDELFIA® wash buffer. Next, 50 μl of DELFIA® Enhancement Solution (Cat#1244-105, Perkin-Elmer, Waltham, Mass.) was added to each wellcontaining cells for a final 15 minute room temperature incubation withshaking. The plates were then read (TRF, excitation 330 nm, emission 620nm) on a SPECTRAMAX® (Molecular Devices, Sunnyvale, Calif.) platereader.

FIGS. 15A-H are representative of the binding curves obtained by thismethod (results are shown for Ab10, Ab20, Ab21, Ab1.H, Ab10.H, Ab21.H,Ab22, and Ab23 respectively) wherein the PAC1-R expressing cells werePC-12 cells. Ab1.H demonstrated binding to the surface of PAC1-Rexpressing cells in the presence of PACAP38, while Ab20, Ab21, Ab10.H,Ab21.H, Ab22, and Ab23 did not appear to appreciably bind to the surfaceof PAC1-R expressing cells using this assay. The binding of Ab1.H to thecell surface of PAC1-R cells was only observed in the presence ofPACAP38. Without intent to be bound by theory, it is hypothesized thatthe binding of the antibodies to the cell surface was mediated bybinding of PACAP38 to GAGs that were present on the cell surface, sincebinding of PACAP38 by GAGs has been previously demonstrated as a PAC1-Rreceptor independent mechanism of PACAP38 binding and internalization byPC-12 cells (see Doan et al., 2012; Juhàsz et al., 2014; and Neree etal., 2015).

Example 7 Inhibition of PACAP38-Induced Dermal Vasodilation in Rabbitsby Anti-PACAP Antibody Ab1.H

Intradermal injection of PACAP38 has been shown to elicit a localizedvasodilation in rabbits and humans (Warren et al., J. Cardio.Pharmacol., 29(1): 83-87, 1992; Seelinger et al., Am. J. Path.,177(5):2563-2575, 2010). An in vivo efficacy study was conducted todetermine the activity of Ab1.H to inhibit a localized dermalvasodilation induced by an intradermal injection of PACAP38 in male NewZealand White rabbits.

Groups of 4 rabbits were dosed with either 90 mg/kg of Ab1.H or withnegative control vehicle (25 mM histidine, 250 mM sorbitol, pH 6.0).Injections were performed by IV (ear vein) bolus administration on day0. Prior to each rabbit PACAP38 challenge, the scapular region of eachanimal was clipped free of hair and wiped with 20% (v/v) alcohol inwater. On day 2, the animals were pre-anesthetized with ketaminehydrochloride and maintained under deep anesthesia with isoflurane gas.Four sites (Region of Interest (“ROI”)) for injection were identified onthe back of each animal using a SHARPIE® permanent marker. Dermalvasodilation and blood perfusion were monitored using the PeriCam PSI NRsystem for Laser Speckle Contrast Analysis (“LASCA”) imaging (Perimed,Järfälla, Sweden), before (baseline) and for 35 minutes afterintradermal PACAP38 challenge. Intradermal PACAP38 challenge wasperformed as follows: each animal received single intradermaladministrations (100 μl/site) of vehicle (one site or ROI) and PACAP38at 30 pmoles/site (3 sites or 3 ROIs). The blood perfusion rates foreach ROI were reported by the PeriCam PSI NR system in Perfusion units(“PU”) and analyzed using PIMSoft (Ver. 1.5, Perimed, Jäsfälla, Sweden).

For each treatment group, the relative % PU change following Ab1.H ornegative control administration compared to baseline was calculated foreach ROI (% PU change for each PACAP38 challenge site−% PU change forthe vehicle site). The relative % PU change in the Ab1.H group wascompared to the relative % PU change in the Negative control group byperforming a two-tailed unpaired t-test statistical evaluation usingGraphPad Prism (version 5.0d, GraphPad Software, La Jolla, Calif.)software.

FIG. 20 demonstrates that Ab1.H inhibited PACAP38-induced dermalvasodilation in rabbits, indicating effectiveness of the antibody atneutralizing PACAP38 activity in vivo.

Example 8 Inhibition of PACAP38-induced Dermal Vasodilation in Rabbitsby Anti-PACAP Antibody Ab10

Intradermal injection of PACAP38 has been shown to elicit a localizedvasodilation in rabbits and humans (Warren et al., 1992; and Seelingeret al., 2010). An in vivo efficacy study was conducted to determine theactivity of Ab10 to inhibit a localized dermal vasodilation induced byan intradermal injection of PACAP38 in male New Zealand White rabbits.

Groups of 4 rabbits were dosed with either 72 mg/kg of Ab10 or withisotype antibody control. Injections were by (ear vein) bolusintravenous administration on day 0. Prior to each rabbit PACAP38challenge, the scapular region of each animal was clipped free of hairand wiped with 20% (v/v) alcohol in water. On day 2, the animals werepre-anesthetized with ketamine hydrochloride and maintained under deepanesthesia with isoflurane gas. Four sites (ROIs) for injection wereidentified on the back of each animal using a SHARPIE® permanent marker.Dermal vasodilation and blood perfusion were monitored using the PeriCamPSI NR system for LASCA imaging (Perimed, Järfälla, Sweden), before(baseline) and for 35 minutes after intradermal PACAP38 challenge.Intradermal PACAP38 challenge was performed as follows: each animalreceived single intradermal administrations (100 μl/site) of vehicle(one site or ROI) and PACAP38 at 30 pmoles/site (3 sites or 3 ROIs). Theblood perfusion rates for each ROI were reported by the PeriCam PSI NRsystem in PU and analyzed using PIMSoft (Ver. 1.5 (Perimed, Järfälla,Sweden)).

For each treatment group, the relative % PU change following Ab10 orIsotype Ab control administration compared to baseline was calculatedfor each ROI (% PU change for each PACAP38 challenge site−% PU changefor the vehicle site). The relative % PU change in the Ab10 group wascompared to the relative % PU change in the Isotype Ab control group byperforming a two-tailed unpaired t-test statistical evaluation usingGraphPad Prism (version 5.0d, GraphPad Software, La Jolla, Calif.)software.

FIG. 21 demonstrates that Ab10 inhibited PACAP38-induced dermalvasodilation in rabbits, indicating effectiveness of the antibody atneutralizing PACAP38 activity in vivo.

Example 9 Epitope Binning of Anti-PACAP Antibodies, Ab1 and Ab10

Ab1 was biotinylated at a 10:1 molar ratio with biotin (Thermo FisherScientific, Waltham, Mass.) per manufacturer guidelines. A 5 stepbiolayer interferometry experiment was performed as follows: In step 1,streptavidin biosensors (Pall ForteBio LLC, Menlo Park, Calif.) wereequilibrated for 50 seconds in 1× kinetics buffer (a 1:10 dilution inDBS of Pall ForteBio LLC, Menlo Park, Calif., cat#18-5032). In step 2, a2 μg/ml dilution of biotinylated antibody Ab1 in 1× kinetics buffer wasimmobilized for 500 seconds onto Streptavidin biosensors. In step 3, theantibody-functionalized biosensors were incubated in a solution of 2 μMunlabeled PACAP peptide (American Peptide Company, Sunnyvale, Calif.,catalog #34-0-20) in 1× kinetics buffer for 200 seconds. In step 4, thesensors were placed into 67 nM solutions of either unlabeled antibodyAb10 (FIG. 22A) or unlabeled antibody Ab1 as control (FIG. 22B) in 1×kinetics buffer for a 1000 second association step. Stability of bindingwas monitored during step 5 for a 1000 second dissociation in 1×kinetics buffer. In FIG. 22A, the “sandwich-style” capture of Ab10 viaAb1-captured PACAP indicates simultaneous and non-competitive binding ofthese two antibodies to PACAP. The control experiment in FIG. 22B showsminimal “sandwich-style” capture of Ab1 via Ab1-captured PACAP. Theexperiment was conducted on a ForteBio OCTET® QK instrument (PallForteBio LLC, Menlo Park, Calif.) at 30° C. and 1000 RPM.

Example 10 Inhibition of PACAP27 Binding to Human PAC1-R by Anti-PA CAPAntibodies

To identify antibodies that block PACAP27 binding to PAC1-R, antibodiesat an initial concentration of 30 nM were diluted in incubation buffer(50 mM Hepes pH 7.4, 1 mM CaCl₂, 5 mM MgCl₂, 0.2% BSA) and serial 1:3dilutions were performed. Antibody dilutions (30 nM, 10 nM, 3 nM, 1 nM,0.3 nM, 0.1 nM, 0.03 nM, 0.01 nM, 0.003 nM and 0.001 nM) were then mixedand pre-incubated at 25° C. for 30 minutes with 0.1 nM of ¹²⁵I-labelledPACAP27 in incubation buffer. The antibody: ¹²⁵I-labelled PACAP27mixture was then added to 0.5 μg aliquots of cell membranes derived fromChem-1 cells expressing human recombinant PAC1-R long isoform inincubation buffer. The mixture was then incubated for 1 hour at 25° C.Following incubation, the samples were filtered and washed. Afterward,the filters were counted to quantitate ¹²⁵I-labelled PACAP27. As anexperimental control, non-specific binding to the cell membranes wasestimated using 0.1 μM of labeled PACAP27. The results indicated thatAb1.H, Ab10.H, and Ab21.H were capable of blocking PACAP27 binding toPAC1-R, thereby demonstrating inhibition of ligand-receptor binding bythe tested antibodies presented in Table 9.

TABLE 9 Inhibition (IC₅₀) of 0.1 nM ¹²⁵I-PACAP27 binding to PAC1-R byanti-PACAP antibodies ANTIBODY IC₅₀ (nM) Ab1.H 0.70 Ab10.H 0.22 Ab21.H0.39

Example 11 Effect of Anti-PACAP Antibody on Light Aversion

To examine the effect of anti-PACAP antibodies on photophobia, a mousemodel was employed in which mice were administered PACAP to triggerphotophobia. Photophobia was detected using a light aversion assay usinga light-dark box as described in Kaiser et al., J. Neurosci.,32(44):15439-15449, 2012. Mice were then administered anti-PACAPantibodies Ab1.H or Ab10.H or an unrelated control antibody and theiraversion to light quantitated. Results are reflected in FIGS. 23-25.

Light Aversion Assay

As described in Kaiser et al., the testing chambers were a plexiglasopen field (27 cm wide×27 cm deep×20.3 cm high) containing three sets of16 beam infrared arrays (two sets of perpendicular beams cross at aheight of 1.0 cm to detect mouse location and locomotion, and the thirdbeam crosses the width of the chamber at a height of 7.3 cm to detectvertical activity). The field was divided in two equal sized zones by adark insert, which is a five-sided, black-colored plexiglas box with atop, but no floor. The use of infrared light beams allowed tracking inboth zones. An opening (5.2 cm×6.8 cm) in the dark insert allowed freemovement between zones. While the dark insert blocked direct light, somelight could still enter through the opening. Each testing chamber waslocated inside a sound-attenuating cubicle (56 cm wide×38 cm deep×36 cmhigh) with a fan for ventilation (Med Associates, Inc.®, St. Albans,Vt.). A computer using Activity Monitor v6.02 (Med Associated Inc.) wasused for recording data from the six chambers.

For each chamber, a LED panel was attached to the ceiling of thesound-attenuating cubicle. The LED panel contains 36 collimated 1 wattLEDs (5500k Daylight White) (LEDwholesalers.com, Burlingame, Calif.). Tocontrol light intensity, each LED panel was connected to a dimmable LEDdriver (LINEARdrive®; eldoLED America Inc., San Jose, Calif.) leading toa potential range of light intensity from 3.0×10² to 2.7×10⁴ 1×. Levelswere further attenuated to 5.5×10¹ 1× using wax paper placed on a clearplexiglass tray below the LEDs. Light intensity was measured withTraceable Dual-Display Light Meter (Control Company, Friendswood, Tex.)placed on the floor of the testing chamber. At 2.7×10⁴ 1×, LED lightsgenerated some heat in the sound attenuating chamber with the dark zoneat ˜25° C. and light zone at ˜27° C.

On the day of the experiment, mice were transported from animal housingand allowed to acclimate to the testing room (˜22° C.) for at least 30to 60 minutes with standard overhead fluorescent lighting (˜200 1×inside the housing cage). Room lights remained on, unless notedotherwise. In addition, all sound-generating equipment were turned onduring acclimation and remained on until testing was complete. There wasminimal human presence in the room during acclimation. Behavioraltesting was performed between 0800 CST and 1400 CST. Any abnormalphysical conditions (e.g. missing eye) were noted.

Ten week old male and female CD1 mice were used in the study (strain#022, Charles River, Wilmington, Mass., US). Mice were allowed torecover from shipping for one to two weeks prior to testing.

Acclimation

All mice were acclimated in the testing room at least 30 to 60 minutesprior to being placed in the light/dark chamber. The light intensity inthe chamber was initially set to 2.7×10³ 1×. The mice were tested forthirty minutes in the chamber every day they were exposed to thelight/dark chamber. Baseline time in light for each mouse was obtainedby exposing the mice to the light/dark chamber twice, with a period ofrest of three days between baseline measurements (FIGS. 23 and 25,“Baseline1” and “Baseline2,” or “Baseline”, respectively).

Treatment

The mice were administered 30 mg/kg of either anti-PACAP antibody orcontrol IgG antibody (negative control antibody having the sameframework as the tested antibodies and that recognizes digoxigenin) byi.p. injection. The mice were then returned to their home cage to restfor one day (24 hours) prior to testing. The mice were then administered0.6 mg/kg PACAP or vehicle by i.p. injection and rested for 30 minutes.The mice were then placed in the light/dark chamber for 30 minutes (FIG.23 and FIG. 25, “Treatment”). After each mouse was exposed to thelight/dark chamber, the light/dark chamber and components were cleanedwith germicidal wipes and dried. About 5 to 7 minutes after a mouse wasplaced in the light/dark chamber, the next mouse to be tested wasinjected with PACAP or vehicle, as described above. This interval wasapproximately the amount of time required to clean the light/darkchamber between experiments.

Motility Measurements

Motility was measured at 5 minute intervals over the 30 minute testingperiod as described in Kaiser et al., J. Neurosci., 2012. Briefly, thenumber of vertical movements, such as rearing, ambulatory distance (cm,the total distance traveled during ambulatory movement status),transitions, and resting (percentage of time spent breaking no newbeams), were measured by light beam. All motility parameters werenormalized to the time spent in each zone to account for differentamount of time spent in that zone; thus, the raw value for eachparameter was divided by the time spent in that zone during the 5 mininterval. Time spent in each chamber was analyzed using GraphPad Prismsoftware (GraphPad Software, San Diego, Calif.), and reported asmean±standard error of the mean (“SEM”). Comparison was calculated bytwo-way repeated measure ANOVA, with Bonferroni's multiple-comparisontest for post-hoc analysis.

Mice were excluded based on three criteria: (1) after the first twoexposures to the box the baseline time in light was analyzed and anymouse that spent +/− one standard deviation of mean time in light atbaseline was removed from the experiment and not given drug treatment,(2) mice were excluded from analysis if they were identified asstatistical outliers (box plot, 10-90%), and (3) mice were excluded ifthey moved less than 10% of the time (combined light and dark).

In two experiments comparing the response of mice administered eitherantibody Ab1.H or Ab10.H to control IgG, the results indicate that miceadministered either PACAP antibody Ab1.H or Ab10.H spent more time inlight as compared to IgG control mice. FIG. 23 shows that mice behavednormally and similarly in both baseline measurements. On the other hand,the data provided in FIG. 23 show that mice treated with control IgGantibody and then PACAP spent statistically less time in light (squares)than mice administered anti-PACAP antibody Ab1.H and then PACAP(circles). (See, FIG. 23, “Treatment”). The data provided in FIG. 25also show that mice behaved normally and similarly in baselinemeasurements. On the other hand, the data provided in FIG. 25 show thatmice treated with control IgG antibody and then PACAP spentstatistically less time in light (triangles) than mice administeredanti-PACAP antibody Ab10.H and then PACAP (inverted triangles). (See,FIG. 25, “Treatment”). Time between each measurement was three days. Themean±SEM is provided for each 5-minute interval. Mice administeredvehicle only behaved as normal controls. Data provided in FIG. 24 showsthat administration of either anti-PACAP antibody Ab1.H, or control IgG,and vehicle (“Veh+PAC Ab” and “Veh+Con Ab,” respectively) did notmarkedly alter mouse behavior. FIG. 24 also shows that the average timeof the mouse in light decreased when PACAP and control IgG wereadministered (“PACAP+Con Ab”), whereas mice administered anti-PACAPantibody Ab1.H and PACAP exhibited normal, non-light-sensitive behavior(“PACAP+PAC Ab”).

Example 12 Epitope Mapping of Anti-PACAP Antibodies

In order to determine the epitopes contained within PACAP to which theanti-PACAP antibodies and antigen binding fragments thereof of theinvention bind, alanine scanning experiments were used. To perform theseexperiments, PACAP peptides were synthesized with a single pointmutation in each position replacing the native amino acid with anAlanine (“Ala”), and the consequences of a single point mutation as itrelates to binding affinity of PACAP and an antibody were measured.Since an alanine residue already occupies positions 18, 24, and 25 ofwild-type PACAP, according to convention, these Ala residues werereplaced with Valine (“Val”) to determine the possible effects of theremoval of the alanine at these positions on the binding of the subjectanti-PACAP antibodies to PACAP. Per the usual convention these Alamutants were labeled according to the position in PACAP 1-38 followed bythe letter code for the substituted amino acid, e.g., 10A indicatesPACAP 1-38 substituted with alanine at amino acid position 10. Bindingof monoclonal antibodies for human PACAP and each mutant peptide wasdetected using SPR on the PROTEON™ XRP36 (Bio-Rad Laboratories,Hercules, Calif.). Samples and sample controls were immobilized onto aPROTEON™ GLC sensor chip (Bio-Rad Laboratories, Hercules, Calif.) at asingle density using standard amine coupling. The running buffer usedfor immobilization was DPBS/modified (HYCLONE™, GE Healthcare LifeSciences, Marlborough, Mass.) and immobilization was conducted at 25° C.The PROTEON™ GLC sensor chip (Bio-Rad Laboratories, Hercules, Calif.)was initialized and pre-conditioned per the manufacturer's protocol(bi-directional injections of 0.5% SDS, 50 mM NaOH, 100 mM HCl). Theimmobilization process was performed step-wise to ensure a uniqueantibody on the spots of the PROTEON™ Chip (Bio-Rad Laboratories,Hercules, Calif.). The surface of the chip was activated with a 1:1mixture of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide(“EDAC/NHS”) and flow rate of 30 μL/min×5 minutes. Antibody samples werepreviously dialyzed or exchanged to 10 mM HEPES 150 mM NaCl pH 7.2, andthe antibody concentration was quantified using a NANODROP®2000spectrophotometer (Thermo Fisher Scientific, Waltham, Mass.). Theimmobilization targeted 2000-3000 response units (“RU”). Antibodysamples (5 μg/ml) in 10 mM sodium acetate, pH 5.5, were flowed at 30μL/min×4 minutes. Deactivation was achieved at a flow rate of 30 μL/minfor 5 minutes using 0.3 M ethanolamine concomitantly with the nextactivation.

Following immobilization, the running buffer was changed to 1× PBST (4.3mM sodium phosphate, 1.4 mM potassium phosphate, 135 mM NaCl, 2.7 mMKCl, 0.05% TWEEN®) with 0.2 M arginine HCl (to reduce non-specificbinding), BSA (0.2 mg/ml, as a carrier) and PROCLIN300® (0.005% as apreservative, Sigma Aldrich, St. Louis, Mo.) and the chip surface wasallowed to re-equilibrate with an injection of new running buffer. Stocksolutions of human PACAP peptide (1-38) and alanine/valine mutantpeptides (Molecular Weight(s): 4.5 kD) at a concentration of 1 mg/mlwere added to the running buffer to final concentrations of 0.45 μg/ml(100 nM). These mixtures were then used to query individual spots on thechip surface with flow rates of 100 μL/min×2 minutes and allowed todissociate for 600 seconds. Chip surfaces were regenerated betweenanalytes by the addition of 0.85% phosphoric acid. Each of antibodiesAb10, Ab20, Ab21, Ab22, and Ab23 were examined under the same conditionsas herein described.

Sensorgrams representing affinity data of mutant peptide binding to apanel of antibodies were assessed using multiple parameters. A visualinspection was first performed for each sensorgram to assess apparentmaximal response (“R_(max)”) relative to the wild-type PACAP peptide(1-38). Second, a visual inspection of the dissociation phase wasperformed with an emphasis on the curve shape relative to the wild-typePACAP peptide. Off-rates (dissociation rates) were calculated forwild-type PACAP peptide and the binding of each mutant peptide to thepanel of antibodies. Finally, as a control experiment to confirm theintegrity of each peptide variant (wild-type or mutant), the bindingaffinity of each member of the peptide library was individuallydetermined for each member of a panel of antibodies that were known tobind wild-type PACAP, to ensure that each Ala mutant PACAP peptideexhibited binding affinity that was similar to the binding affinity ofwild-type PACAP peptide. Collective assessment of all describedparameters identified PACAP amino acid residues important forPACAP/antibody binding.

Binding and dissociation data are shown in FIGS. 26A-30B for binding ofantibodies Ab10, Ab20, Ab21, Ab22, and Ab23 to wild-type PACAP and PACAPmutants. The upper panel in each figure contains the binding data forresidues in PACAP that appeared to be important for antibody binding(labeled at the right end of the graph, e.g., “10A” indicates thebinding data for the mutant containing alanine at position 10 of PACAP).The lower panel provides data points representing the degree of bindingof the remaining PACAP alanine mutants, i.e., PACAP alanine mutants thatbound to the tested antibody similar to wild-type PACAP. Based thereon,the residue was determined to likely not be important for antibodybinding. As a positive control, both the upper and lower panels for eachFigure also disclose the binding data obtained using wild-type PACAP(labeled huPACAP(1-38)).

FIG. 31A-B summarizes the PACAP residue positions determined tocontribute to antibody binding affinity based on data obtained in thesealanine scanning studies. The positions listed in each column identifythe PACAP alanine scanning mutants whose mutation led to a decrease inPACAP/antibody binding affinity. The residue positions are listed incolumn 3 of FIG. 31A-B according to the spatial arrangement of theresidues along the PACAP primary sequence (from amino acid residue1-38). The PACAP residue positions contributing most to antibody bindingwere interpreted to jointly comprise the epitopes bound by eachantibody. Based on data obtained in these alanine scanning studies, theepitopes bound by each antibody were concluded to comprise the followingresidues:

(i) Ab10: residues 19, 22, 23, and 27 of human PACAP.(ii) Ab20: residues 19, 22, 23, 24, and 27 of human PACAP.(iii) Ab21: residues 19, 22, 23, and 27 of human PACAP.(iv) Ab22: residues 22, 23, 27, 28, and 31 of human PACAP.(v) Ab23: residues 12, 20, 23, 24, 26, 27, and 28 of human PACAP.

It was further noted based on the alanine scanning experimental resultsthat the affinity of each of antibodies Ab10, Ab20, Ab21, Ab22, and Ab23for PACAP involves or depends on residues 19, 22, 23, and/or 27 of humanPACAP.

Additionally, it was observed that the affinity of each of antibodiesAb22 and Ab23 to PACAP involves or requires specific amino acid residuesthat are present in human wild-type PACAP38, but which are not presentin human wild-type PACAP27, e.g., residues 28 and 31 of PACAP38.

With respect to the foregoing alanine scanning results humanizedvariants of the subject anti-PACAP antibodies should interact with theidentical or substantially identical residues of human PACAP ashumanization should not appreciably impact the specificity of thebinding of the humanized anti-PACAP antibody to human PACAP compared tothe parent (unhumanized) antibody. Particularly, Ab10.H should interactwith the same residues on human PACAP as Ab10, and Ab21.H shouldinteract with the same residues on human PACAP as Ab21.

Antibodies which bind to the same or overlapping epitopes on human PACAPas the subject antibodies may be produced and identified using methoddescribed herein. It is reasonable to anticipate that antibodies whichbind to the same or overlapping epitope as any of the antibodiesidentified herein will likely possess similar biological activity absenta meaningful difference in binding kinetics. Particularly, suchantibodies should antagonize one or more of the biological effectselicited by PACAP analogously to the exemplified anti-PACAP antibodieswhich bind these epitopes. Additionally, antibodies that bind to thesesame or overlapping epitopes, or a subset of residues thereof, areanticipated to mimic the binding characteristics of the subjectantibodies. For example such antibodies are expected to selectively bindto PACAP and not bind or bind with much less affinity (weaker) to VIP orother peptides within this family of neuropeptides.

Having fully described and enabled the invention, the invention isfurther described by the claims that follow.

1-191. (canceled)
 192. A human, humanized or chimerized anti-humanPituitary Adenylate Cyclase-Activating Polypeptide (“PACAP”) antibody orantibody fragment selected from the following: (i) that antagonizes,inhibits, neutralizes or blocks at least one biological effectassociated with human PACAP; (ii) one that that specifically competesfor binding to human PACAP with an antibody selected from the groupconsisting of Ab10 and Ab20 or an antigen-binding fragment of any one ofthe foregoing; (iii) one that specifically binds to at least one linearor conformational epitope bound by an anti-PACAP antibody selected fromthe group consisting of Ab10 and Ab20 or an antigen-binding fragment ofany one of the foregoing; (iv) one which specifically binds to anepitope on human PACAP or a fragment or variant thereof containing thecorresponding amino acid residues wherein said epitope is selected fromthe group consisting of: (a) at least one of residues 19, 22, 23 and 27of human PACAP; (b) at least one of residues 19, 22, 23, 24 and 27 ofhuman PACAP; (c) at least two of the residues of any one of (i)-(ii);(d) at least three of the residues of any one of (i)-(ii); (e) at leastfour of the residues of any one of (i)-(ii); and (f) all five of theresidues of (ii). (v) which specifically binds to an epitope on humanPACAP, or a fragment or variant thereof containing the correspondingamino acid residues that includes one or more of residues 19, 22, 23 and27 of human PACAP; (vi) one which specifically binds to an epitope onhuman PACAP or a fragment or variant thereof containing thecorresponding amino acid residues, wherein said epitope consists of theresidues (a) or (b) as set forth in (v); (vii) one which specificallybinds to an epitope on human PACAP, or a fragment or variant thereofcontaining the corresponding amino acid residues that includes one ormore of residues 19, 22, 23 and 27 of human PACAP; (viii) one whichspecifically binds to an epitope on human PACAP or a fragment or variantthereof containing the corresponding amino acid residues, wherein saidepitope consists of the residues (a) or (b) as set forth in (v); (ix)which specifically binds to an epitope on human PACAP, or a fragment orvariant thereof containing the corresponding amino acid residues thatincludes one or more of residues 19, 22, 23 and 27 of human PACAP; (x)which specifically binds to an epitope on human PACAP or a fragment orvariant thereof containing the corresponding amino acid residues,wherein said epitope consists of the residues (i) or (ii) as set forthin (v); (xi) the antibody or fragment of any of the foregoing whichspecifically binds to an epitope on human PACAP or a fragment or variantthereof containing the corresponding amino acid residues that is presentin human wild-type PACAP38 and in human wild-type human PACAP27; (xii)the antibody or fragment of any of the foregoing which specificallybinds to an epitope on human PACAP (or a fragment or variant thereofcontaining the corresponding amino acid residues) wherein said epitopeis identified by alanine scanning, e.g., as disclosed in Example 12;(xiii) which does not bind to or does not appreciably bind to humanVasoactive Intestinal Peptide (“VIP”); (xiv) which has a K_(D) for humanPACAP which is at least 10, 100, 1000, 10,000 or 100,000 fold lower(stronger) than the K_(D) of said antibody or antibody fragment to humanVIP; (xv) which inhibits or neutralizes at least one biological effectelicited by human PACAP; (xvi) which comprises one or more of thefollowing: a. inhibits, blocks or prevents PACAP activation of at leastone of PAC1 receptor (“PAC1-R”), vasoactive intestinal peptide receptortype 1 (“VPAC1-R”), and/or vasoactive intestinal peptide receptor type 2(“VPAC2-R”); b. inhibits, blocks or prevents PACAP activation of each ofPAC1-R, VPAC1-R, and VPAC2-R; c. inhibits, blocks or prevents PACAPactivation of PAC1-R; d. is capable of inhibiting PACAP binding to atleast one of PAC1-R, VPAC1-R, and/or VPAC2-R; e. is capable ofinhibiting PACAP binding to each of PAC1-R, VPAC1-R, and/or VPAC2-R; f.is capable of inhibiting PACAP binding to PAC1-R-expressing cells; g. iscapable of inhibiting PACAP binding to VPAC1-R-expressing cells; h. iscapable of inhibiting PACAP binding to VPAC2-R-expressing cells; i.inhibits PACAP binding to the cell surface, e.g. via a glycosaminoglycan(“GAG”), j. inhibits PACAP-mediated binding of such antibody to the cellsurface, e.g., via a glycosaminoglycan (“GAG”); k. inhibits, blocks orprevents PACAP-induced cAMP production; and/or l. when administered to asubject reduces PACAP-induced vasodilation (xvii) which is substantiallynon-immunogenic in human subjects; (xviii) is suitable for treating ahuman subject having an acute, episodic or chronic condition associatedwith increased vasodilation, mast cell degranulation and/or neuronalactivation; (xix) which specifically binds to the same or overlappinglinear or conformational epitope(s) and/or competes for binding to thesame or overlapping linear or conformational epitope(s) on human PACAPas an anti-PACAP antibody selected from Ab10 or Ab20; (xx) whichspecifically binds to the same or overlapping linear or conformationalepitope(s) on human PACAP as an anti-PACAP antibody selected from Ab10or Ab20; (xxi) the epitope bound by said antibody is identified byalanine scanning, e.g., as disclosed in Example 12; (xxii) it comprisesat least 2, 3, 4, 5 or all 6 complementarity determining regions(“CDRs”) of an anti-PACAP antibody selected from Ab10 or Ab20,preferably the V_(H) CDR3 and V_(L) CDR3; (xxiii) it comprises asequence variant of any of the foregoing containing one or moremodifications that putatively alter binding affinity or immunogenicity;(xxiv) it comprises: a. a variable heavy chain comprising the CDR1sequence consisting of SEQ ID NO: 404; a CDR2 sequence consisting of SEQID NO: 406; and a CDR3 sequence consisting of SEQ ID NO: 408; and/or b.a variable light chain comprising the CDR1 sequence consisting of SEQ IDNO: 424; a CDR2 sequence consisting of SEQ ID NO: 426; and a CDR3sequence consisting of SEQ ID NO: 428; (xxv) it comprises: (a) avariable heavy chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 402,and/or (b) a variable light chain comprising an amino acid sequence withat least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO: 422; (xxvi) it comprises: (a) a variable heavy chain having theamino acid sequence of SEQ ID NO: 402, and/or (b) a variable light chainhaving the amino acid sequence of SEQ ID NO: 422; (xxvii) it comprises:(a) a heavy chain having the amino acid sequence of SEQ ID NO: 401,and/or (b) a light chain having the amino acid sequence of SEQ ID NO:421; (xxviii) it comprises: (a) a variable heavy chain comprising theCDR1 sequence consisting of SEQ ID NO: 444; a CDR2 sequence consistingof SEQ ID NO: 446; and a CDR3 sequence consisting of SEQ ID NO: 448;and/or (b) a variable light chain comprising the CDR1 sequenceconsisting of SEQ ID NO: 464; a CDR2 sequence consisting of SEQ ID NO:466; and a CDR3 sequence consisting of SEQ ID NO: 468; (xxix) itcomprises: (a) a variable heavy chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO: 442, and/or (b) a variable light chain comprising an aminoacid sequence with at least 80, 85, 90, 95, 96, 97, 98, or 99% sequenceidentity to SEQ ID NO: 462; (xxx) it comprises: (a) a variable heavychain having the amino acid sequence of SEQ ID NO: 442, and/or (b) avariable light chain having the amino acid sequence of SEQ ID NO: 462;(xxxi) it comprises: (a) a heavy chain having the amino acid sequence ofSEQ ID NO: 441, and/or (b) a light chain having the amino acid sequenceof SEQ ID NO: 461; (xxxii) it is selected from the group consisting ofscFvs, camelbodies, nanobodies, Immunoglobulin New Antigen Receptor(“IgNAR”), fragment antigen binding (“Fab”) fragments, Fab′ fragments,MetMab like antibodies, monovalent antigen binding fragments, andF(ab′)₂ fragments; (xxxiii) it substantially or entirely lacksN-glycosylation and/or O-glycosylation; (xxxiv) it comprises a humanconstant domain; (xxxv) it is an IgG1, IgG2, IgG3, or IgG4 antibody;(xxxvi) it comprises an Fc region that has been modified to alter atleast one of effector function, half-life, proteolysis, orglycosylation; (xxxvii) it comprises an Fc region containing one or moremutations that alters or eliminates N- and/or O-glycosylation; (xxxviii)the antibody or antigen binding fragment binds to PACAP with a bindingaffinity (K_(D)) of less than or equal to 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M,10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M,10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, or 10⁻¹³ M,e.g., as determined by ELISA, bio-layer interferometry (“BLI”), KINEXAor surface plasmon resonance at 25° or 37° C.; (xxxix) the anti-PACAPantibody or antigen binding fragment binds to PACAP with a bindingaffinity (KD) of less than or equal to 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M,10⁻¹¹ M, 5×10⁻¹²M, or 10⁻¹² M; (xl) the antibody or antigen bindingfragment binds to PACAP with an off-rate (k_(off)) of less than or equalto 5×10⁻⁴ s⁻¹, 10⁻⁴ s⁻¹, 5×10⁻⁵ s⁻¹, or 10⁻⁵ s⁻¹; (xli) the antibody orantigen binding fragment is directly or indirectly attached to adetectable label or therapeutic agent; (xlii) the antibody or antigenbinding fragment when administered to a subject inhibits or neutralizesat least one biological effect elicited by PACAP; (xliii) the anti-PACAPantibody or antibody fragment binds to PACAP27 and/or PACAP38 and blocksPACAP27 and/or PACAP38 binding to PAC1-R, VPAC1-R, and/or VPAC2-R;(xliv) the anti-PACAP antibody or antibody fragment binds to PACAP27and/or PACAP38 and blocks PACAP27 and/or PACAP38 binding to each ofPAC1-R, VPAC1-R, and VPAC2-R; (xlv) the anti-PACAP antibody or antibodyfragment of any of the foregoing which binds to PACAP27 and/or PACAP38and blocks PACAP27 and/or PACAP38 binding to PAC1-R-expressing cells,VPAC1-R-expressing cells, and/or VPAC2-R-expressing cells; (xlvi) theanti-PACAP antibody or antibody fragment of any of the foregoing whichneutralizes or inhibits PACAP activation of at least one of PAC1-R,VPAC1-R, or VPAC2-R; (xlvii) the anti-PACAP antibody or antibodyfragment of any of the foregoing which neutralizes or inhibits PACAPactivation of each of PAC1-R, VPAC1-R, and VPAC2-R; (xlviii) theanti-PACAP antibody or antibody fragment of any of the foregoing whichneutralizes or inhibits PACAP activation of PAC1-R; (xlix) theanti-PACAP antibody or antibody fragment of any of the foregoing whichis capable of inhibiting PACAP binding to at least one of PAC1-R,VPAC1-R, or VPAC2-R; (l) the anti-PACAP antibody or antibody fragment ofany of the foregoing which is capable of inhibiting PACAP binding toeach of PAC1-R, VPAC1-R, and VPAC2-R; (li) the anti-PACAP antibody orantibody fragment of any of the foregoing which is capable of inhibitingPACAP binding to PAC1-R-expressing cells, VPAC1-R-expressing cells,and/or VPAC2-R-expressing cells; (lii) the anti-PACAP antibody orantibody fragment of any of the foregoing which inhibits PACAP-inducedcAMP production; (liii) the anti-PACAP antibody or antibody fragment ofany of the foregoing which when administered to a subject reducesPACAP-induced vasodilation, mast cell degranulation and/or neuronalactivation; (liv) the anti-PACAP antibody or antibody fragment of any ofthe foregoing which binds to PACAP with a K_(D) that is less than about100 nM; (lv) the anti-PACAP antibody or antibody fragment of any of theforegoing which binds to PACAP with a K_(D) that is less than about 40nM. (lvi) the anti-PACAP antibody or antibody fragment of any of theforegoing which binds to PACAP with a K_(D) that is less than about 100pM; (lvii) the anti-PACAP antibody or antibody fragment of any of theforegoing which binds to PACAP with a K_(D) that is less than about 50pM; (lviii) the anti-PACAP antibody or antibody fragment of any of theforegoing which binds to PACAP with a K_(D) that is less than about 25pM; (lix) the anti-PACAP antibody or antibody fragment of any of theforegoing which binds to PACAP with a KD that is between about 10 pM andabout 100 pM; (lx) the anti-PACAP antibody or antibody fragment of anyof the foregoing which has stronger affinity for PACAP as compared toVIP and/or does not bind to VIP; (lxi) the anti-PACAP antibody orantibody fragment of any of the foregoing wherein the affinity of saidantibody or antigen binding fragment to PACAP is at least 10-fold,30-fold, 100-fold, 300-fold, 1000-fold, 3000-fold, 10000-fold,30000-fold, 100000-fold, 300000-fold, 1000000-fold, 3000000-fold,10000000-fold, 30000000-fold or more stronger than the affinity of saidantibody or antigen binding fragment to VIP; (lxii) the anti-PACAPantibody or antibody fragment of any of the foregoing wherein theantibody or antigen binding fragment is attached to at least oneeffector moiety, e.g., a chemical linker; (lxiii) the anti-PACAPantibody or antibody fragment of any of the foregoing wherein theantibody or antigen binding fragment is attached to one or moredetectable moieties, e.g., comprises a fluorescent dye, enzyme,substrate, bioluminescent material, radioactive material,chemiluminescent moiety, or mixtures thereof; (lxiv) the anti-PACAPantibody or antibody fragment of any of the foregoing wherein theantibody or antigen binding fragment is attached to one or morefunctional moieties; (lxv) an anti-idiotypic antibody specific to ananti-PACAP antibody or antibody fragment of any of the foregoing, whichoptionally, neutralizes one or more biological effects of the anti-PACAPantibody to which it binds.
 193. A method of using the anti-idiotypicantibody of claim 192 to monitor the in vivo levels of said anti-PACAPantibody or antigen binding fragment in a subject or to neutralize invivo effects of said anti-PACAP antibody in a subject.
 194. Acomposition suitable for therapeutic, prophylactic or a diagnostic usecomprising a therapeutically, prophylactically or diagnosticallyeffective amount of at least one anti-PACAP antibody or antigen bindingfragment or anti-idiotypic antibody according to claim
 192. 195. Thecomposition of claim 194 which comprises at least one of the following:is administrable via injection, topical, oral, inhalation or transdermaladministration; or is suitable for subcutaneous administration; issuitable or for intravenous administration; it is lyophilized; itfurther comprises a pharmaceutically acceptable diluent, carrier,solubilizer, emulsifier, preservative, or mixture thereof; it comprisesanother active agent; it comprises another active agent selected fromthe group consisting of a chemotherapeutic, an analgesic, ananti-inflammatory, an immunosuppressant, a cytokine, anantiproliferative, an antiemetic, and a cytotoxin; and/or it islyophilized, stabilized, and/or formulated for administration byinjection.
 196. An isolated nucleic acid sequence or nucleic acidsequences encoding an anti-PACAP antibody or antigen binding fragment oranti-idiotypic antibody according to claim 192 or a vector or vectorscontaining.
 197. A host cell comprising the isolated nucleic acidsequence or sequences of claim 196 or the vector or vectors of claim196, optionally a mammalian, bacterial, fungal, yeast, avian, amphibian,plant or insect cell; or is a filamentous fungus or a yeast or is amammalian cell; or yeast is selected from the following genera:Arxiozyma; Ascobotryozyma; Citeromyces; Debaryomyces; Dekkera;Eremothecium; Issatchenkia; Kazachstania; Kluyveromyces; Kodamaea;Lodderomyces; Pachysolen; Pichia; Saccharomyces; Saturnispora;Tetrapisispora; Torulaspora; Williopsis; and Zygosaccharomyces or aPichia cell; or a Pichia is selected from Pichia pastoris, Pichiamethanolica or Hansenula polymorpha (Pichia angusta), or is afilamentous fungus or a yeast or is a CHO cell.
 198. A method ofexpressing an anti-PACAP antibody or antigen binding fragment comprisingculturing the host cell of claim 197, optionally a yeast or mammaliancell, e.g., a CHO cell under conditions that provide for expression ofsaid antibody or antigen binding fragment; optionally the host cell is ayeast or CHO cell that stably expresses and secretes into the culturemedium said antibody or antigen binding fragment; or a polyploid yeast,optionally said polyploid yeast, optionally a Pichia yeast is made by amethod that comprises: (i) introducing at least one expression vectorcontaining one or more heterologous polynucleotides encoding saidantibody operably linked to a promoter and a signal sequence into ahaploid yeast cell; (ii) producing by mating or spheroplast fusion apolyploid yeast from said first and/or second haploid yeast cell; (iii)selecting polyploid yeast cells that stably express said antibody; and(iv) producing stable polyploid yeast cultures from said polyploid yeastcells that stably express said antibody into the culture medium.
 199. Amethod of blocking, inhibiting, blocking or neutralizing one or morebiological effects associated with PACAP in a subject comprisingadministering to said subject a therapeutically or prophylacticallyeffective amount of a human, humanized or chimerized anti-PACAP antibodyor antibody fragment that antagonizes, inhibits, neutralizes or blocksat least one biological effect associated with human PACAP; preferablyan antibody according to claim
 192. 200. The method according to claim199 wherein the human, humanized or chimerized anti-PACAP antibody orantibody fragment comprises one or more of the following: i. inhibits,blocks or neutralizes at least one biological effect elicited by PACAP;ii. neutralizes or inhibits PACAP activation of at least one of PAC1receptor (“PAC1-R”), vasoactive intestinal peptide receptor type 1(“VPAC1-R”), and/or vasoactive intestinal peptide receptor type 2(“VPAC2-R”); iii. inhibits, blocks or neutralizes PACAP activation ofeach of PAC1-R, VPAC1-R, and VPAC2-R; (d) neutralizes or inhibits PACAPactivation of PAC1-R; iv. is capable of inhibiting PACAP binding to atleast one of PAC1-R, VPAC1-R, and/or VPAC2-R; v. is capable ofinhibiting PACAP binding to each of PAC1-R, VPAC1-R, and/or VPAC2-R; vi.is capable of inhibiting PACAP binding to PAC1-R-expressing cells; vii.is capable of inhibiting PACAP binding to VPAC1-R and/orVPAC2-R-expressing cells; viii. inhibits PACAP binding to the cellsurface, e.g., via a glycosaminoglycan (“GAG”); ix. inhibitsPACAP-mediated binding of such antibody to the cell surface, e.g., viaGAG; x. inhibits PACAP-induced cyclic adenosine monophosphate (“cAMP”)production; and/or xi. when administered to the subject reducesPACAP-induced vasodilation, mast cell degranulation and/or neuronalactivation.
 201. The method of claim 199 that blocks inhibits, orneutralizes vasodilation, e.g., vasodilation of the dural arteries,which is associated with or elicited by pituitary adenylatecyclase-activating peptide (“PACAP”) e.g., that blocks, inhibits, orneutralizes vasodilation associated with, or elicited by PACAP or whichtreating or preventing the onset, frequency, severity or duration ofheadache or migraine in a subject; optionally headache or migraine isselected from migraine with aura, migraine without aura, hemiplegicmigraine, cluster headache, migrainous neuralgia, chronic headache,chronic migraine, medication overuse headache, and tension headache; orwhich treats a human subject having an acute, episodic or chroniccondition associated with at least one of increased vasodilation, mastcell degranulation and neuronal activation or a combination of any ofthe foregoing.
 202. The method of claim 199, wherein the subject has acondition selected from the group consisting of migraine with aura,migraine without aura, hemiplegic migraines, cluster headaches,migrainous neuralgia, chronic headaches, tension headaches, generalheadaches, hot flush, chronic paroxysmal hemicrania, secondary headachesdue to an underlying structural problem in the head, secondary headachesdue to an underlying structural problem in the neck, cranial neuralgia,sinus headaches, headache associated with sinusitis, allergy-inducedheadaches, allergy-induced migraines, trigeminal neuralgia,post-herpetic neuralgia, phantom limb pain, fibromyalgia, reflexsympathetic dystrophy, pain, chronic pain, inflammatory pain,post-operative incision pain, post-surgical pain, trauma-related pain,lower back pain, eye pain, tooth pain, complex regional pain syndrome,cancer pain, primary or metastatic bone cancer pain, fracture pain,osteoporotic fracture pain, pain resulting from burn, gout joint pain,pain associated with sickle cell crises, pain associated withtemporomandibular disorders, cirrhosis, hepatitis, neurogenic pain,neuropathic pain, nociceptic pain, visceral pain, menstrual pain,ovarialgia, osteoarthritis pain, rheumatoid arthritis pain, diabeticneuropathy, sciatica, dyspepsia, irritable bowel syndrome, inflammatorybowel disease, Crohn's disease, ileitis, ulcerative colitis, renalcolic, dysmenorrhea, cystitis, interstitial cystitis, menstrual period,labor, menopause, pancreatitis, schizophrenia, depression,post-traumatic stress disorder (“PTSD”), anxiety disorders, autoimmunediabetes, Sjögren's syndrome, multiple sclerosis, overactive bladder,bronchial hyperreactivity, asthma, stroke, bronchitis, bronchodilation,emphysema, chronic obstructive pulmonary disease (“COPD”), inflammatorydermatitis, adenocarcinoma in glandular tissue, blastoma in embryonictissue of organs, carcinoma in epithelial tissue, leukemia in tissuesthat form blood cells, lymphoma in lymphatic tissue, myeloma in bonemarrow, sarcoma in connective or supportive tissue, adrenal cancer,AIDS-related lymphoma, anemia, bladder cancer, bone cancer, braincancer, breast cancer, carcinoid tumors, cervical cancer, chemotherapy,colon cancer, cytopenia, endometrial cancer, esophageal cancer, gastriccancer, head cancer, neck cancer, hepatobiliary cancer, kidney cancer,leukemia, liver cancer, lung cancer, lymphoma, Hodgkin's disease,non-Hodgkin's, nervous system tumors, oral cancer, ovarian cancer,pancreatic cancer, prostate cancer, rectal cancer, skin cancer, stomachcancer, testicular cancer, thyroid cancer, urethral cancer, cancer ofbone marrow, multiple myeloma, tumors that metastasize to the bone,tumors infiltrating the nerve and hollow viscus, tumors near neuralstructures, acne vulgaris, atopic dermatitis, urticaria, keloids,hypertrophic scars and rosacea, endothelial dysfunction, Raynaud'ssyndrome, coronary heart disease (“CHD”), coronary artery disease(“CAD”), heart failure, peripheral arterial disease (“PAD”), diabetes,pulmonary hypertension (“PH”), connective tissue disorder, allergicdermatitis, psoriasis, pruritus, neurogenic cutaneous redness, erythema,sarcoidosis, shock, sepsis, opiate withdrawal syndrome, morphinetolerance, and epilepsy; and/or the subject has a condition selectedfrom the group consisting of migraine, headache and a pain associateddisease or condition; and/or the subject has a the headache or migraineis selected from the group consisting of migraine with aura, migrainewithout aura, hemiplegic migraine, cluster headache, migrainousneuralgia, chronic headache, chronic migraine, medication overuseheadache, and tension headache; and/or the subject has a ocular disorderassociated with photophobia selected from the group consisting ofachromatopsia, aniridia, photophobia caused by an anticholinergic drug,aphakia, buphthalmos, cataracts, cone dystrophy, congenitalabnormalities of the eye, viral conjunctivitis, corneal abrasion,corneal dystrophy, corneal ulcer, disruption of the corneal epithelium,ectopia lentis, endophthalmitis, eye trauma caused by disease, eyetrauma caused by injury, eye trauma caused by infection, chalazion,episcleritis, glaucoma, keratoconus, optic nerve hypoplasia,hydrophthalmos, congenital glaucoma iritis, optic neuritis, pigmentdispersion syndrome, pupillary dilation, retinal detachment, scarring ofthe cornea, sclera and uveitis; and/or the subject has a nervoussystem-related or neurological condition associated with photophobiaselected from the group consisting of autism spectrum disorders, Chiarimalformation, dyslexia, encephalitis, meningitis, subarachnoidhemorrhage, tumor of the posterior cranial fossa, ankylosingspondylitis, albinism, ariboflavinosis, benzodiazepines, chemotherapy,chikungunya, cystinosis, Ehlers-Danlos syndrome, hangover, influenza,infectious mononucleosis, magnesium deficiency, mercury poisoning,migraine, rabies, and tyrosinemia type II; and/or the subject has aphotophobia associated disorder selected from the group consisting ofmigraine with aura, migraine without aura, iritis, uveitis, meningitis,depression, bipolar disorder, cluster headache or anther trigeminalautonomic cephalalgia (“TAC”) or blepharospasm, depression, agoraphobia,and bipolar disorder.
 203. The method of claim 199 that furthercomprises one or more of the following: the method further comprisesadministering separately or co-administering another agent; optionallythe other agent is selected from a chemotherapeutic, an analgesic, ananti-inflammatory, an immunosuppressant, a cytokine, anantiproliferative, an antiemetic or a cytotoxin; optionally the othertherapeutic agent is an analgesic; optionally a non-steroidalanti-inflammatory drug (“NSAID”), an opioid analgesic, another antibodyor a non-antibody biologic; optionally an anti-NGF antibody or antigenbinding fragment, further optionally an anti-Calcitonin Gene-RelatedPeptide (“CGRP”) antibody or antigen binding fragment; furtheroptionally the NSAID is a cyclooxygenase 1 and/or cyclooxygenase 2inhibitor.
 204. An anti-PACAP antibody or antibody fragment according toclaim 192 comprising one or more of the following: it inhibits theeffects of PACAP on vasodilation; it inhibits the effects of PACAP oncAMP production; inhibits the effects of PACAP on PLC resulting inreduced Ca++ and PLD levels; it inhibits the effects of PACAP onadenylate cyclase activity; it inhibits the effects of PACAP on itsbinding to any or all of PAC1-R, VPAC1-R or VPAC2-R; it inhibits theeffects of PACAP on neurodevelopment it inhibits the effects of PACAP onneuroprotection; which inhibits the effects of PACAP on neuromodulation;inhibits the effects of PACAP on neurogenic inflammation; it inhibitsthe effects of PACAP on nociception; it modulates the interaction ofPACAP with the cell surface, e.g., via interaction with at least oneglycosaminoglycan (“GAG”); e.g., one or more of heparin, chondroitin,keratin, and hyaluronic acid; it blocks or inhibits receptor-independentcellular uptake of PACAP38 and/or PACAP27 and/or it inhibits or blocksGAG-dependent uptake of PACAP38 and/or PACAP27 by cells.
 205. A methodaccording to claim 199 wherein the administered anti-PACAP antibody orantibody fragment comprises one or more of the following: it inhibitsthe effects of PACAP on vasodilation; it inhibits the effects of PACAPon cAMP production; it inhibits the effects of PACAP on PLC resulting inreduced Ca++ and PLD levels; it inhibits the effects of PACAP onadenylate cyclase activity; it inhibits the effects of PACAP on itsbinding to any or all of PAC1-R, VPAC1-R or VPAC2-R; it inhibits theeffects of PACAP on neurodevelopment; which inhibits the effects ofPACAP on neuroprotection; it inhibits the effects of PACAP onneuromodulation; it inhibits the effects of PACAP on neurogenicinflammation; it inhibits the effects of PACAP on nociception; itmodulates the interaction of PACAP with the cell surface, e.g., viainteraction with at least one glycosaminoglycan (“GAG”); e.g., one ormore of heparin, chondroitin, keratin, and hyaluronic acid; it blocks orinhibits receptor-independent cellular uptake of PACAP38 and/or PACAP27;and/or it inhibits or blocks GAG-dependent uptake of PACAP38 and/orPACAP27 by cells.
 206. The method of claim 199 that further comprisesthe administration of one or more of the following: another active agentis selected from a chemotherapeutic, an analgesic, an anti-inflammatory,an immunosuppressant, a cytokine, an antiproliferative, an antiemetic ora cytotoxin; another active agent selected from an opioid analgesic,another antibody or a non-antibody biologic; e.g., an anti-NGF oranti-CGRP antibody; or an NSAID comprising a cyclooxygenase 1 and/orcyclooxygenase 2 inhibitor; or the NSAID is selected from the groupconsisting of (1) propionic acid derivatives including ibuprofen,naproxen, naprosyn, diclofenac, and ketoprofen; (2) acetic acidderivatives including tolmetin and sulindac; (3) fenamic acidderivatives including mefenamic acid and meclofenamic acid; (4)biphenylcarboxylic acid derivatives including diflunisal and flufenisal;and (5) oxicams including piroxim, sudoxicam, and isoxicam; or opioidanalgesic is selected from the group consisting of codeine,dihydrocodeine, diacetylmorphine, hydrocodone, hydromorphone,levorphanol, oxymorphone, alfentanil, buprenorphine, butorphanol,fentanyl, sufentanil, meperidine, methadone, nalbuphine, propoxyphene,pentazocine, and pharmaceutically acceptable salts thereof; or an opioidanalgesic comprising morphine or a morphine derivative orpharmaceutically acceptable salt thereof, optionally wherein the opioidanalgesic and the anti-PACAP antibody or antigen binding fragmentincrease the analgesic effect as compared to either the opioid analgesicor the anti-PACAP antibody or antigen binding fragment administeredalone; optionally in combination with another active agent that elicitsa synergistic or additive effect on the treatment or prevention of aPACAP associated effect, e.g., migraine or on pain.